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Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5.

A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spotswere visualized, and 181 protein spotswere characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same proteinmay be detected in two ormore neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks. ispartof: MicrobiologyOpen vol:1 issue:2 pages:169-181 ispartof: location:England status: published

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.

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.

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.

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.

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.

Summary No single fly gonadotropin has as yet been identified with certainty. Dababase analysis fails to yield positive results when the genomic database of Drosophila is scanned for the presence of family members of gonadotropins that have already been identified in other insects. However, a candidate gonadotropic peptide (cAMP generating peptide, Neb-cGP) was purified some time ago from the fleshfly Neobellieria bullata. A short form of this peptide was found to stimulate cAMP production in the ovaries of the fleshfly. In the present study, we gathered additional arguments that suggest a role for Neb-cGP as a gonadotropic hormone. Brains of male and female adult Neobellieria bullata were stained with antiserum directed to the cAMP generating peptide. In both sexes four lateral neurosecretory cells in the dorsal part of each pars lateralis were stained. In addition, mass spectrometric read-outs of extracts of fleshfly corpora cardiaca displayed the full length form (48 amino acid residues) of the cAMP-ge...

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 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.

Postgenomic, precursor, expressed sequence tag, and mass spectrometric peptidomic analyses allowed us to identify a single gene leading to a novel 18mer-isoform of a bety-type pigment-dispersing ho ...