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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.

The honeybee has an invaluable economic impact and is a model for studying immunity, development and social behavior. The recent sequencing and annotation of the honeybee genome facilitates the study of its hemolymph, which reflects the physiological condition and mediates immune responses. We aimed at making a proteomic reference map of honeybee hemolymph and compared gel-free and gel-based techniques. One hundred and four 2-DE spots corresponding to 62 different proteins were identified. Eight identical 2-DLC experiments resulted in the identification of 32 unique proteins. One repeat was clearly not representative for the potential of the given 2-DLC setup. Only 27% of the identified hemolymph proteins were found by both techniques. In addition, we found proteins of three different viruses which creates possibilities for biomarker design. Future hemolymph studies will benefit from this work.

This review is focussed on SIFamide. This neuropeptide was discovered as a result of an extensive purification process, typical for 20th century physiology, of an extract of 350,000 flesh flies. Our knowledge of SIFamide greatly expanded since the first publication in 1996. Describing the minor and major findings on this peptide is our lead to summarise a number of innovations that recently became common in research on Arthropods. Mass spectrometry, nanoLC, whole mount immunocytochemistry, genome sequencing, deorphanizing receptors and functional gene knock downs are aspects that dramatically improved and changed peptide research. Some of the techniques mentioned in this review were of course applied before 1996, but they were not widespread. Although the focus of the review is on insects we incorporated the data of SIFamide in Crustaceans as well. SIFamide illustrates that crustaceans and insects might have more in common than was previously anticipated. Today, six isoforms of SIFamide are discovered in many crustaceans, several insects and a tick. The sequence of SIFamide is extremely conserved among these species. Deorphanizing its receptor in Drosophila, learned that both the ligand and receptor are impressively conserved, pointing at a crucial function. Immunohistochemistry and mass spectrometry data reveal that SIFamide is present in the crustacean brain and gut, but restricted to four neurons in the insect pars intercerebralis. The immunoreactive patterns in the brain refer to a neuromodulatory role in combining visual, tactile and olfactory input. Eventually, targeted cell ablation and RNAi revealed that SIFamide modulates sexual behaviour in fruit flies.

The neuropeptide-like precursor 1 (NPLP1) was first identified in a peptidomics experiment on Drosophila melanogaster. Limited data on this novel neuropeptide precursor suggest a role in the regulation of ecdysis in holometabolous larvae. In this study, we characterized the NPLP1 precursor in the gray flesh fly, Neobellieria bullata, which is an excellent model for physiological assays and hence to discover the role of the NPLP1 peptides. Antisera against three of the D. melanogaster NPLP1 neuropeptides stained an identical set of neurons in the central nervous system of N. bullata compared to D. melanogaster. A novel approach was applied to identify the N. bullata NPLP1 orthologs. Using a combination of affinity chromatography, mass spectrometry, cDNA cloning and RACE experiments, we obtained almost the complete coding sequence of the NPLP1 mRNA. Three encoded NPLP1 peptides were identified in central nervous system extracts by mass spectrometry. Neither doses of 25-250pmol of synthetic Neb-MGYamide and Neb-PQNamide peptides, nor the NPLP1 antisera did affect the speed of retraction, contraction and tanning in the pupariation bioassay.

Insulin is an extensively studied peptide hormone in mammals. However, insulin is not restricted to vertebrates, but has also been identified in invertebrates, among whom several insect species. These insulin-like peptides (ILPs) show structural and-at least some-functional homology with mammalian insulin and act through a conserved pathway. Yet many aspects of insulin function in insects remain to be unveiled. We analyzed the presence of ILPs in the cotton leafworm, Spodoptera littoralis, at two levels: (1) cellular localization of ILPs in whole tissues of the central nervous system from S. littoralis, and (2) detection and identification of ILPs at nucleotide level. To our knowledge, nothing about the presence of ILPs in S. littoralis has been described so far. By whole mount in situ immunolocalization, we localized bombyxin-like material in S. littoralis in four pairs of pars intercerebralis cells and in the corpus cardiacum-corpus allatum complexes. In addition, we have cloned two different S. littoralis ILP precursor cDNAs by a combination of PCR and RAcE. The corresponding precursor polypeptides ('Sl-ILPP1' and 'Sl-ILPP2') show significant sequence homology with precursors for bombyxin and other bombyxin-related peptides. Our results strongly suggest that the S. littoralis ILPs belong to the category of bombyxin-analogs. ispartof: General and Comparative Endocrinology vol:153 issue:1 pages:72-9 ispartof: location:ENGLAND, Univ Sheffield, Sheffield status: published

Antimicrobial peptides (AMPs) play an important role in the innate immunity of insects. In Drosophila 17 additional immune induced molecules (DIMs) were found in the haemolymph of adult flies upon septic injury. Previous studies using MALDI mass spectrometry combined with Edman degradation, detected AMPs and DIMs of a predominantly large size. By means of 2D-nanoLC ESI MS/MS, 43 DIMs were identified in this study from the haemolymph of Drosophila third instar larvae 12 h after challenge with a mixture of Micrococcus luteus and Escherichia coli. Most peptides were derived from known AMP or DIM precursors, but only four peptides were purified and identified before. The majority of the peptides that we detected were smaller in size. Interestingly, two previously unknown peptide precursors were found and hereby related to immune defense. These include CG7738 and CG32185. Many of the identified peptides are post-translationally modified by an N-terminal pyroglutamic acid and/or a C-terminal amide. Haemolymph of control larvae was treated in the same way and revealed only one peptide.

Bioactive peptides play critical roles in regulating most biological processes in animals. The elucidation of the amino acid sequence of these regulatory peptides is crucial for our understanding of animal physiology. Most of the (neuro)peptides currently known were identified by purification and subsequent amino acid sequencing. With the entire genome sequence of some animals now available, it has become possible to predict novel putative peptides. In this way, BLAST (Basic Local Alignment Searching Tool) analysis of the Drosophila melanogaster genome has allowed annotation of 36 secretory peptide genes so far. Peptide precursor genes are, however, poorly predicted by this algorithm, thus prompting an alternative approach described here. With the described searching program we scanned the Drosophila genome for predicted proteins with the structural hallmarks of neuropeptide precursors. As a result, 76 additional putative secretory peptide genes were predicted in addition to the 43 annotated ones. These putative (neuro)peptide genes contain conserved motifs reminiscent of known neuropeptides from other animal species. Peptides that display sequence similarities to the mammalian vasopressin, atrial natriuretic peptide, and prolactin precursors and the invertebrate peptides orcokinin, prothoracicotropic hormones, trypsin modulating oostatic factor, and Drosophila immune induced peptides (DIMs) among others were discovered. Our data hence provide further evidence that many neuropeptide genes were already present in the ancestor of Protostomia and Deuterostomia prior to their divergence. This bioinformatic study opens perspectives for the genome-wide analysis of peptide genes in other eukaryotic model organisms.

Here, we report the identification of proteins associated with the immune response of Drosophila by analyzing the hemolymph profiles after infection. Two-dimensional difference gel electrophoresis was used to study the secretome in the hemolymph of Drosophila larvae. Shortly after induction with lipopolysaccharides, we identified 10 proteins, which we designated "Drosophila instantly released immune proteins". Infection with Micrococcus luteus or Saccharomyces cerevisiae induced 20 and 19 differential protein spots, respectively. Next to known immune proteins, new candidates that require further investigation were identified.

Peptides are the largest class of signalling molecules found in animals. Nevertheless, in most proteomic studies peptides are overlooked since they literally fall through the mazes of the net. In analogy with proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole pepticlome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications. In this paper we describe the analysis of the larval fruit fly central nervous system using two-dimensional capillary liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS/MS. Using the central nervous systems of only 50 larval Drosophila as starting material, we identified 38 peptides in a single analysis, 20 of which were not detected in a previous study that reported on the one-dimensional capillary LC/MS/MS analysis of the same tissue. Among the 38 sequenced peptides, some originate from precursors, such as the tachykinin and the IFamide precursor that were entirely missed in the first study. This clearly demonstrates that the two-dimensional capillary LC approach enhances the coverage of the peptidomic analysis. Copyright (C) 2005 John Wiley Sons, Ltd.

Neb-LFamide or AYRKPPFNGSLFamide was originally purified from the grey flesh fly Neobellieria bullata as a myotropic neuropeptide. We studied the occurrence of this peptide and its isoforms in the central nervous system of different insect species by means of whole mount fluorescence immunohistochemistry, mass spectrometry, and data mining. We found that both sequence and immunoreactive distribution pattern are very conserved in the studied insects. In all species and stages we counted two pairs of immunoreactive cells in the pars intercerebralis. These cells projected axons throughout the ventral nerve cord. In the adult CNSs they formed a large number of immunoreactive varicosities as well. Mass spectrometry and data mining revealed that SIFamide exists in two isoforms: [G1]-SIFamide and [A1]-SIFamide. In addition, the SIFamide joining peptide is relatively well conserved throughout arthropod species. The conserved presence of two cysteine residues, separated by six amino acid residues, allows the formation of disulphide bridges.

Peptides from the pyrokinin/PBAN family and some structurally related compounds identified in various arthropods were tested for acceleration of puparial contraction in flesh fly larvae. Modifications of behavioural patterns of pupariation were further studied for the active compounds using a behavioural analysis based on the recording of changes in tension of the cuticle. Nine peptides belonging to the pyrokinin/PBAN family (Lem-PK, Pea-PK-5, Lom-PK II, Hez-PBAN, Bom-DH-I), identified in five different insect species, two pyrokinin peptides derived from the genome of Drosophila melanogaster (capa-3, and hugin), and two pyrokinins identified from the white shrimp Penaeus vannamei were very active in the pupariation assay, with threshold doses within the range of 0.1-5.0 pmol larva(-1). High activity was also detected for a related peptide ETH1 from Drosophila. All of these peptides share a C-terminal PRLamide, which is essential and sufficient for the activity. Interestingly, two other structurally related peptides from Drosophila--ETH2 and capa-1--which feature conservative changes (Ile and Val, respectively) at the C-terminal Leu position, were inactive within a physiological range of concentrations. It is clear that the receptor mediating the acceleration of puparial contraction behaviour is sensitive to the introduction of greater steric bulk at the C-terminal Leu position. The peptides that accelerated pupariation showed very similar patterns of muscular and cuticular activity.

Insects respond to microbial infection by the rapid and transient expression of several genes encoding antibacterial peptides. In this paper we describe a powerful technique, two-dimensional difference gel electrophoresis, that, when combined with mass spectrometry, can be used to study the immune response of Drosophila melanogaster at the protein level. By comparatively analyzing the hemolymph proteome of 2,000 third-instar Drosophila larvae, we identified 10 differential proteins that appear in the fruit fly hemolymph very early after an immune-challenge with lipopolysaccharides. These proteins can be assigned to the immune response, because they are not induced after sterile injury. Reduction of intergel variability or quantification problems related to conventional two-dimensional electrophoresis and improvement of image analysis were achieved by the use of two fluorescent dyes to label the two different protein samples. Some of the immune-induced proteins, such as thioester-containing protein 2, can be assigned to specific aspects of the immune response; others were already reported as being involved in stress response. An immune-induced protein (CG18594) is homologous to a mammalian serine protease inhibitor that mediates the mitogen-activated protein kinase and the NF-κB signaling pathways. In addition, a number of proteins that had not been associated with the immune response before were isolated and identified, and some of these were still present in the hemolymph 4 h after injury. Determining the function of all of these immune-induced proteins represents an exciting challenge for increasing our knowledge of insect immunity.

The pars intercerebralis-corpora cardiaca complex in insects is the functional equivalent of the vertebrate brain-pituitary axis. During the past few decades more than 40 neuropeptides have been isolated from the locust brain-corpus cardiacum complex. Tedious and time-consuming successive purification rounds of large tissue extracts were necessary to achieve the purification and sequencing of most of these signal molecules. Nowadays, the combination of nanoscale liquid chromatography and the very sensitive tandem mass spectrometry allows us to identify and sequence peptides in very low concentration directly from tissue extracts. In this manuscript, we review previous data on the peptidome analysis of the locust corpora cardiaca, with emphasis on AKH processing. In addition, we report the peptide profiling of a single corpus cardiacum from Locusta migratoria. 23 peptides were isolated and sequenced in a single nano-LC-MS/MS experiment, demonstrating the sensitivity and effectiveness of mass spectrometry in peptide research.

Eusocial insect societies display a remarkable reproductive division of labor between a single fertile queen and thousands of largely sterile workers. In most species, however, the workers retain the capacity to reproduce, particularly in queenless colonies where typically many workers lay eggs. As yet, the molecular determinants that initiate this shift in worker fertility are still poorly documented. By using RNA interference we here demonstrate that the knockdown of epidermal growth factor receptor, a gene which was previously shown to be involved in queen-worker caste differentiation, also induces reproduction in worker honeybees (Apis mellifera). These data show that worker fertility and queen-worker caste determination partly rely on the same gene regulatory networks, thereby providing a major breakthrough in our understanding of the molecular determinants of the social insects’ spectacular reproductive division of labor.

Eusocial behavior is extensively studied in the honeybee, Apis mellifera, as it displays an extreme form of altruism. Honeybee workers are generally obligatory sterile in a bee colony headed by a queen, but the inhibition of ovary activation is lifted upon the absence of queen and larvae. Worker bees are then able to develop mature, viable eggs. The detailed repressive physiological mechanisms which are responsible for this remarkable phenomenon are as yet largely unknown. Physiological studies today mainly focus on the transcriptome, while the proteome stays rather unexplored. Here, we present a quantitative 2-dimensional differential gel electrophoresis comparison between activated and inactivated worker ovaries and brains of reproductive and sterile worker bees, including a spot map of ovaries, containing 197 identified spots. Our findings suggest that suppression of ovary activation might involve a constant interplay between primordial oogenesis and subsequent degradation, which is probably mediated through steroid and neuropeptide hormone signaling. Additionally, the observation of higher viral protein loads in both the brains and ovaries of sterile workers is particularly noteworthy. This dataset will be of great value for future research unraveling the physiological mechanisms underlying the altruistic sterility in honeybee workers. ispartof: Journal of Proteome Research vol:11 issue:5 pages:2838-2850 ispartof: location:United States status: published

A defining feature of social insects is the reproductive division of labour, in which workers usually forego all reproduction to help their mother queen to reproduce. However, little is known about the molecular basis of this spectacular form of altruism. Here, we compared gene expression patterns between nonreproductive, altruistic workers and reproductive, non-altruistic workers in queenless honeybee colonies using a whole-genome microarray analysis. Our results demonstrate massive differences in gene expression patterns between these two sets of workers, with a total of 1292 genes being differentially expressed. In nonreproductive workers, genes associated with energy metabolism and respiration, flight and foraging behaviour, detection of visible light, flight and heart muscle contraction and synaptic transmission were overexpressed relative to reproductive workers. This implies they probably had a higher whole-body energy metabolism and activity rate and were most likely actively foraging, whereas same-aged reproductive workers were not. This pattern is predicted from evolutionary theory, given that reproductive workers should be less willing to compromise their reproductive futures by carrying out high-risk tasks such as foraging or other energetically expensive tasks. By contrast, reproductive workers mainly overexpressed oogenesis-related genes compared to nonreproductive ones. With respect to key switches for ovary activation, several genes involved in steroid biosynthesis were upregulated in reproductive workers, as well as genes known to respond to queen and brood pheromones, genes involved in TOR and insulin signalling pathways and genes located within quantitative trait loci associated with reproductive capacity in honeybees. Overall, our results provide unique insight into the molecular mechanisms underlying alternative reproductive phenotypes in honeybee workers.

The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers. ispartof: PLoS One vol:6 issue:6 pages:7- ispartof: location:United States status: published

The striking similarities between the innate defences of vertebrates and invertebrates as well as the amenability of Caenorhabditis elegans for genetic analysis, have made this free-living ground nematode a popular model system in the study of bacterial pathogenesis. Although genetic studies have brought new insights, showing the inducibility and pathogen-specificity of the immune response, there is still much to be discovered about the exact mechanisms underlying resistance to infection. In this paper a different angle was adopted to study host-pathogen interactions in C. elegans. We report the application of differential gel electrophoresis (DIGE), combined with mass spectrometry to search for proteins that are differentially synthesised in the worm after infection with the gram-negative bacterium Aeromonas hydrophila. Given the dynamic nature of an immune response, the proteome of C. elegans was investigated at three different time-points after infection. A total of 65 differential proteins were identified. This study confirms the involvement of galectins, C-type lectins and lipid binding proteins in the immunity of C. elegans. In addition a number of unknown proteins, which might represent important players of the worm's defence system, were isolated and identified. This work gives a first indication of the complex changes that occur at the protein level during infection.

Corazonin (Crz) is an 11 amino acid C-terminally amidated neuropeptide that has been identified in most arthropods examined with the notable exception of beetles and an aphid. The Crz-receptor shares sequence similarity to the GnRH-AKH receptor family thus suggesting an ancestral function related to the control of reproduction and metabolism. In 1989, Crz was purified and identified as a potent cardioaccelerating agent in cockroaches (hence the Crz name based on "corazon", the Spanish word for "heart"). Since the initial assignment as a cardioacceleratory peptide, additional functions have been discovered, ranging from pigment migration in the integument of crustaceans and in the eye of locusts, melanization of the locust cuticle, ecdysis initiation and in various aspects of gregarization in locusts. The high degree of structural conservation of Crz, its well-conserved (immuno)-localization, mainly in specific neurosecretory cells in the pars lateralis, and its many functions, suggest that Crz is vital. Yet, Crz-deficient insects develop normally. Upon reexamining all known effects of Crz, a hypothesis was developed that the evolutionary ancient function of Crz may have been "to prepare animals for coping with the environmental stressors of the day". This function would then complement the role of pigment-dispersing factor (PDF), the prime hormonal effector of the clock, which is thought "to set a coping mechanism for the night".

The honeybee is the economically most important beneficial insect and a model for studying immunity, development and social behavior. Hence, this species was selected for genome sequencing and annotation. An intensive interplay between bioinformatics and mass spectrometry (MS) resulted in the annotation of 36 neuropeptide genes (Hummon et al., 2006). Exactly 100 peptides were demonstrated by a variety of MS techniques. In this follow-up study we dissected and analysed separately all ganglia of the central nervous system (CNS) of adult worker bees in three repeats. The combined MALDI-TOF spectra enabled the accurate mapping of 67 peptides, encoded by 20 precursors. We also demonstrated the expression of an additional but already predicted peptide. In addition to putative bioactive peptides we also list and discuss spacer peptides, propeptides and truncated peptides. The majority of such peptides have a more restricted distribution pattern. Their presence provides some information on the precursor turnover and/or the location of neural cell bodies in which they are produced. Of a given precursor, the (neuro)-peptides with the widest distribution pattern are likely to be the best candidates to interact with receptors. The separate analysis of a neuroendocrine complex and the mushroom body yields suggestions as to which (neuro)-peptides might act as hormones and which neuropeptides might be involved in the complex spectrum of non-hormone driven honeybee behaviour, at these sites. Our data complement immunohistochemical studies of (neuro)-peptides in the honeybee, and form a reference for comparative studies in other insect or arthropod models, in particular in the light of recent or upcoming genome projects. Finally, they also form a firm basis for physiological, functional and/or differential peptidomics studies in the honeybee.

Background For holometabolous insects there has been an explosion of proteomic and peptidomic information thanks to large genome sequencing projects. Heterometabolous insects, although comprising many important species, have been far less studied. The migratory locust Locusta migratoria, a heterometabolous insect, is one of the most infamous agricultural pests. They undergo a well-known and profound phase transition from the relatively harmless solitary form to a ferocious gregarious form. The underlying regulatory mechanisms of this phase transition are not fully understood, but it is undoubtedly that neuropeptides are involved. However, neuropeptide research in locusts is hampered by the absence of genomic information. Results Recently, EST (Expressed Sequence Tag) databases from Locusta migratoria were constructed. Using bioinformatical tools, we searched these EST databases specifically for neuropeptide precursors. Based on known locust neuropeptide sequences, we confirmed the sequence of several previously identified neuropeptide precursors (i.e. pacifastin-related peptides), which consolidated our method. In addition, we found two novel neuroparsin precursors and annotated the hitherto unknown tachykinin precursor. Besides one of the known tachykinin peptides, this EST contained an additional tachykinin-like sequence. Using neuropeptide precursors from Drosophila melanogaster as a query, we succeeded in annotating the Locusta neuropeptide F, allatostatin-C and ecdysis-triggering hormone precursor, which until now had not been identified in locusts or in any other heterometabolous insect. For the tachykinin precursor, the ecdysis-triggering hormone precursor and the allatostatin-C precursor, translation of the predicted neuropeptides in neural tissues was confirmed with mass spectrometric techniques. Conclusion In this study we describe the annotation of 6 novel neuropeptide precursors and the neuropeptides they encode from the migratory locust, Locusta migratoria. By combining the manual annotation of neuropeptides with experimental evidence provided by mass spectrometry, we demonstrate that the genes are not only transcribed but also translated into precursor proteins. In addition, we show which neuropeptides are cleaved from these precursor proteins and how they are post-translationally modified.

Ce travail résulte de la collaboration de très nombreux chercheurs. Seuls les auteurs de la rubrique Physical and Genetic Mapping sont cités explicitement.; Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A1T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A.mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A.mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement

A brain-retrocerebral complex-subesophageal ganglion acidified methanolic extract of 100 larval Galleria mellonella (greater wax moth) was prepared for the isolation and identification of (neuro)peptides. To reduce sample complexity, the isolated peptides were roughly separated using a single, conventional chromatographic separation step. Subsequently, screening of these fractions with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in combination with nanoflow electrospray ionization quadrupole time-of-flight tandem mass spectrometry resulted in the identification of 12 lepidopteran peptides. None of these had been previously isolated or characterized within this species. VIFTPKLamide encoded by the diapause hormone-pheromone biosynthesis activating neuropeptide precursor was for the first time isolated and biochemically identified in a tissue extract, providing irrefutable evidence of its expression in larval nervous tissue. Another pentapeptide, AMVRFamide, with no resemblance to other lepidopteran peptides, was de novo sequenced and is most related to the neuropeptide F peptide family.

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.

In this study, we analyzed the hemolymph proteome of Drosophila third instar larvae, which were induced with a suspension of Gram-positive bacteria or yeast. Profiling of the hemolymph proteins of infected versus non-infected larvae was performed by two-dimensional difference gel electrophoresis. Infection with Micrococcus luteus or Saccharomyces cerevisiae induced, respectively, 20 and 19 differential protein spots. The majority of the spots are specifically regulated by one pathogen, whereas only a few spots correspond to proteins altered in all cases of challenging (including after challenge with lipopolysaccharides). All of the upregulated proteins can be assigned to specific aspects of the immune system, as they did not increase in the hemolymph of sterile pricked larvae. Next to known immune proteins, unannotated proteins were identified such as CG4306 protein, which has homologues with unknown function in all metazoan genome databases available today.

Thirty-five years ago, Zdarek and Fraenkel demonstrated that nervous tissue extracts influenced development by accelerating pupariation in the grey flesh fly, Neobellieria bullata. We have now identified this pupariation factor as SVQFKPRLamide, designated Neb-pyrokinin-2 (Neb-PK-2). To achieve this, the central nervous system of N. bullata wandering stage larvae, that is, preceding pupariation, were dissected and extracted before HPLC separation. Chromatographic fractions were screened with a bioassay for pupariation accelerating activity. Only one fraction showed huge pupariation activity. Mass spectrometry revealed the presence of a pyrokinin, whose primary sequence could not be unequivocally determined by tandem mass spectrometry. However, this Neb-pyrokinin appeared to be very prominent in the ring gland from which it was subsequently purified and identified. Synthetic Neb-PK-2 accelerates pupariation with a threshold dose of only 0.2 pmol and therefore, Neb-pyrokinin is considered to be the genuine pupariation factor. The immunohistochemical distribution pattern of Neb-PK-2 is very similar to that of Drosophila pyrokinin-2, from which it differs by only one amino acid residue. Hence, the recently identified G-protein coupled receptors (CG8784, CG8795) for Drosophila pyrokinin-2 might play an important role in puparium formation.

A peptidomics approach was applied to determine the peptides in the larval central nervous system of the grey flesh fly, Neobellieria bullata. Fractions obtained by high performance liquid chromatography were analysed by MALDI-TOF and ESI-Q-TOF mass spectrometry. This provided biochemical evidence for the presence of 18 neuropeptides, 11 of which were novel Neobellieria peptides. Most prominently present were the FMRFamide-related peptides: 7 FMRFamides, 1 FIRFamide, and Neb-myosuppressin. The three putative capa-gene products Neb-pyrokinin and the periviscerokinins Neb-PVK-1 and -2 were detected, as well as another pyrokinin. This Neb-PK-2 was also present in the ring gland along with corazonin, Neb-myosuppressin, and Neb-AKH-GK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained Neb-LFamide, proctolin, and FDFHTVamide, designated as Neb-TVamide. With this study, we considerably increased our knowledge of the neuropeptidome of the pest fly N. bullata, which is an important insect model for physiological research.

Advances in mass spectrometry and the availability of genomic databases made it possible to determine the peptidome or peptide content of a specific tissue. Peptidomics by nanoflow capillary liquid chromatography tandem mass spectrometry of an extract of 50 larval Drosophila brains, yielded 28 neuropeptides. Eight were entirely novel and encoded by five not yet annotated genes; only two genes had a homologue in the Anopheles gambiae genome. Seven of the eight peptides did not show relevant sequence homology to any known peptide. Therefore, no evidence towards the physiological role of these 'orphan' peptides was available. We identified one of the eight peptides, IPNamide, in an extract of the Drosophila adult brain as well. Next, specific antisera were raised to reveal the distribution pattern of IPNamide and other peptides from the same precursor, in larval and adult brains by means of whole-mount immunocytochemistry and confocal microscopy. IPNamide immunoreactivity is abundantly present in both stages and a striking similarity was found between the distribution patterns of IPNamide and TPAEDFMRFamide, a member of the FMRFamide peptide family. Based on this distribution pattern, IPNamide might be involved in phototransduction, in processing sensory stimuli, as well as in controlling the activity of the oesophagus.

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis -activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 μg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 μg/0.2 g fresh wt). Cry1E and Cry1Ac (5 μg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 μg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 μg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.