Neuropeptidomics of the grey flesh fly, Neobellieria bullata

BBRC Biochemical and Biophysical Research Communications 316 (2004) 763–770 www.elsevier.com/locate/ybbrc

Neuropeptidomics of the grey flesh fly, Neobellieria bullata Peter Verleyen,* Jurgen Huybrechts, Filip Sas, Elke Clynen, Geert Baggerman, Arnold De Loof, and Liliane Schoofs Laboratory of Developmental Physiology, Genomics and Proteomics, K.U.Leuven, Naamsestraat 59, Louvain B-3000, Belgium Received 3 February 2004

Abstract 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-AKHGK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained NebLFamide, 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. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Neobellieria bullata; Peptidomics; Mass spectrometry; FMRFamide-related peptides; Pyrokinins; Periviscerokinins

The recent publications of the genomes from the fruit fly Drosophila melanogaster [1] and the malaria mosquito Anopheles gambiae [2] have stirred a major revolution in insect research. They enabled the straightforward identification of proteins and peptides that play a key role in the endocrinology and physiology of insects. We are interested in neuropeptides and their receptors since they occupy a high hierarchical position in the physiology of insects and regulate most (if not all) biological processes, such as reproduction, metamorphosis, feeding, homeostasis, and behaviour. Recently, peptide profiling of the Drosophila central nervous system (CNS) has led to the isolation and sequencing of 28 neuropeptides in a single peptidomic experiment [3]. These results were confirmed by an immunocytochemical approach, demonstrating the cellular localisation of the newly identified Drosophila peptide IPNamide in the larval CNS [4]. In addition, about 20 neuropeptide Gprotein coupled receptors have been identified and characterised in Drosophila using reverse pharmacology * Corresponding author. Fax: +32-16-32-39-02. E-mail address: [email protected] (P. Verleyen).

0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.115

or orphan receptor strategies [5–9]. While Drosophila is especially suited for functional genomic and proteomic studies, the related and larger-sized dipteran pest species, Neobellieria bullata (formerly called Sarcophaga bullata), remains more appropriate for conducting physiological experiments. The progress that has been made with respect to neuropeptide identification in Drosophila will be very useful for the identification of biologically active neuropeptides in the closely related grey flesh fly. In fact, to date, only 11 neuropeptides have been characterised in N. bullata, including Neb-LFamide [10], neomyosuppressin [11], neosulfakinins [12], Neb-kinin [13], NebAKH [14], Neb-FMRFamide and Neb-FIRFamide [8], and Neb-periviscerokinins and Neb-pyrokinin [15], although immunocytochemical data suggest the presence of more peptides [16–18]. Advances in mass spectrometry have made it possible to identify a substantial number of neuropeptides in nervous tissue, even in species without genomic sequence information like Locusta migratoria and Cancer borealis [19,20]. Nevertheless, determining the peptidome of a species without an available genomic database of a related species

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continues to be a difficult task. The present study describes the identification of 18 neuropeptides, 11 of which are novel N. bullata peptides, from an extract of the central nervous system of wandering stage larvae.

Materials and methods Insects, tissue extraction, and pre-purification. N. bullata was reared as described [21]. The central nervous systems of 700 wandering stage larvae were collected and homogenised in a solution of methanol/ water/acetic acid (90:9:1 v/v/v) immediately upon dissection. After sonication, the homogenate was centrifuged at 9000g for 10 min. The pellet was resuspended in extraction medium and again sonicated and centrifuged. Both supernatants were pooled. After evaporation of the methanol, the sample was delipidated using ethyl acetate and n-hexane. A Sep-Pak C18 cartridge (Waters) was used for solid phase extraction. The loaded and washed cartridge was eluted with 60% acetonitrile (CH3 CN), containing 0.1% trifluoroacetic acid (TFA). After evaporation, the remaining aqueous sample was filtered through a Millipore 0.22 lm filter, prior to HPLC analysis. To identify the peptidome of the ring gland, 10 ring glands from wandering stage larvae were dissected, extracted, sonicated, and centrifuged. The supernatant was completely evaporated and then the sample was redissolved in 50 ll of 0.1% TFA in water. A ZipTipC18 (Millipore, 15 lm) was used to concentrate and desalt the sample. The sample was eluted with 5 ll CH3 CN/water/formic acid (FA) (70:29.9:0.1 v/v/v). One millilitre was analysed by MALDI-TOF MS, two microliter was analysed by Q-TOF MS/MS. High performance liquid chromatography and capillary LC (CapLC). High performance liquid chromatography (HPLC) analysis was performed on a Gilson liquid chromatograph. The Waters 486 Tunable Absorbance detector was set at 214 nm for peptide detection. The separation was carried out on a Symmetry Prep C8 column (7.8
300 mm, 7 lm) with a flow rate of 2 ml/min. After injection of the sample, containing an equivalent of 700 central nervous systems, a 1-h linear gradient from 2% to 60% solvent B was imposed [A: water/ TFA (99.9:0.1 v/v); B: CH3 CN/TFA (99.9:0.1 v/v)]. Fractions were collected every minute. An equivalent of 7 larval central nervous systems from every fraction was lyophilised and resuspended in 5 ll water/ CH3 CN/formic acid (70:29.9:0.1 v/v/v) for MALDI-TOF analysis. An equivalent of 100 central nervous systems was treated the same way for Q-TOF analysis.

Mass spectrometry with MALDI-TOF and Q-TOF. Matrix-assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (MS) was performed on a Reflex IV (Bruker, Germany), equipped with a N2 laser and pulsed ion extraction accessory. The instrument was calibrated using a standard peptide mixture (Bruker, Germany) as described [22]. Final spectra resulted from 20 to 100 shots, recorded in the reflectron mode within a mass range from m=z 500 to m=z 3000. MALDI-TOF MS was used to screen the ring gland extract and the HPLC fractions of the central nervous system. Each time, only 1 ll was transferred to a ground steel target plate, mixed with 0.5 ll saturated a-cyano-4-hydroxycinnamic acid in ethanol/ CH3 CN/TFA (50:49.9:0.1 v/v/v), and air-dried. Nanoflow electrospray ionisation (ESI) double quadrupole (Qq) orthogonal acceleration (oa) time-of-flight (TOF) mass spectrometry (MS) was performed on a Q-TOF system (Micromass, UK). Q-TOF MS was used to analyse the ring gland extract as well as the chromatographic fractions obtained from the central nervous system extract. Each time, only 2 ll of the prepared water/CH3 CN/FA solution was loaded into a gold-coated capillary (Long NanoES spray capillaries, Proxeon Biosystems A/S, Denmark). The sample was sprayed at a flow rate of 30 nl/min. During tandem MS, fragment ions are generated from a selected precursor ion by collision-induced dissociation (CID). The collision energy is typically varied between 20 and 35 V, in order to fragment the parent ion into a satisfying number of different daughter ions. The amino acid sequence is determined by calculating the m=z difference (which corresponds to the mass of an amino acid residue) between the adjacent y-ion peaks and/or b-ion peaks. Masslynx software version 3.5 was used for sequence analysis.

Results and discussion The HPLC separation of 700 larval central nervous systems (CNSs) yielded a satisfying separation of components (Fig. 1). Aliquots containing the equivalent of only 2 and 40 CNSs were consumed for analysis by means of MALDI-TOF MS and Q-TOF MS/MS, respectively, remaining material was used elsewhere. Screening of the HPLC fractions with MALDI-TOF MS revealed a huge number of peptide masses. Only the masses that could be unequivocally linked to a peptide

Fig. 1. HPLC fractionation of an extract, originating from 700 Neobellieria larval central nervous systems, on a Symmetry Prep C8 column (7.8
300 mm, particle size 7 lm). Linear gradient from 2% to 60% B in 1 h (A: 99.9% MilliQ water/0.1% TFA; B: 99.9% CH3 CN/0.1% TFA) with a flow rate of 2 ml/min. UV detector set at 214 nm, absorption units full scale (AUFS) is 1. Fractions were collected every minute, their numbers correspond to the elution time.

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Table 1 Peptides identified by mass spectrometry in HPLC fractions of a larval central nervous system extract from N. bullata Peptide name

Peptide sequence

Molecular mass (monoisotopic) Measured (Da)

Theoretical (Da)

Fraction

Non-amidated peptides Proctolin Neb-AKH-GK

RYLPT pQLTFSPDWGK

648.30 1160.65

648.36 1160.55

23 29

FMRFamide-related peptides Neb-FMRFamide-2

TPSQDFMRFamide

1126.65 1126.63 1209.69 1113.63 1239.50 1149.56 971.57

1126.52

30 31 30 30 33 29 30

Neb-FMRFamide-3 Neb-FMRFamide-4 Neb-FMRFamide-5 Neb-FMRFamide-6 Neb-FMRFamide-7 Neb-FMRFamide-8 Neb-FIRFamide-1 Neb-MS

SPPSQDFMRFamide SANQDFMRFamide LSPTQDFMRFamidea TPTHDFMRFamide EEDFMRFamide Or GGKMFMRFamide AGQDNFMRFamide APPQPSDNFIRFamide TDVDHVFLRFamide

Periviscerokinins and pyrokinins Neb-PVK-1 Neb-PVK-2

NGGTSGLFAFPRVamidea AGLLVYPRLamidea

Neb-PK-1 Neb-PK-2

AGPSATTGVWFGPRLamidea XXXFXPRLamidea

Neb-TVamide Conserved peptides Neb-LFamide Neb-Cor

1083.63 1386.81 1246.55

1209.56 1113.50 1239.61 1149.54 971.42 971.48 1083.49 1386.70 1246.65

30 31 33

1320.69 999.62

FDFHTVamide

1320.60 999.73 999.71 1514.76 972.60 972.59 763.43

763.37

34 31 32 23 25 26 14

AYRKPPFNGSLFamide pQTFQYSRGWTNamide

1394.85 1368.63

1394.75 1368.62

31 29

1514.80 972.59

Peptide names, sequences, and molecular masses (measured by MALDI-TOF MS) are given. Peptides in bold were sequenced using Q-TOF MS/ MS, underlined peptides were identified for the first time in Neobellieria. a Conservative substitution of Leu to Ile cannot be ruled out due to mass ambiguity for this amino acid pair.

sequence are represented (Table 1). Most sequences were verified by Q-TOF MS/MS (Figs. 2–4) except for six peptide sequences. These were identified based on their mass in combination with additional information. For instance, proctolin elutes at the same retention time as the fraction that contains the mass of 648.3 Da, typical for proctolin. The mass readout of chromatographic fraction 29 (corresponding to the elution time of Argcorazonin) of the CNS displays a prominent ion peak of 1368.6 corresponding to the mass of [Arg7 ]-corazonin. Finally, Neb-AKH-GK, Neb-PVK-1, Neb-PVK-2 and Neb-PK-1 were already identified in N. bullata nervous tissue [14,15]. Therefore, ion peaks with exactly the same mass (0.1 Da) as these four peptides definitely correspond to these neuropeptides. MALDI-TOF analysis of an equivalent of only two ring glands showed 16 ion peaks potentially corresponding to peptides (data not shown). Nine masses were also present in HPLC fractions of the larval CNS, four of which could be unmistakably identified as NebAKH-GK, [Arg7 ]-corazonin, Neb-myosuppressin, and the FXPRLamide of fraction 25 (Table 1, Fig. 5). The latter 2 were confirmed by Q-TOF analysis of four ring gland equivalents.

Non-amidated peptides Not surprisingly, most identified peptides were N-terminally amidated, except for proctolin and NebAKH-GK. Proctolin was the first neuropeptide to be isolated and sequenced from insects [23]. It was subsequently found throughout arthropods in motorneurons stimulating a variety of muscles [24]. Recently, the gene for a proctolin preprohormone (CG7105) as well as the gene for a G protein-coupled receptor for proctolin (CG6986) were identified in Drosophila [6,25,26]. NebAKH-GK, an intermediate processing product of the amidated adipokinetic hormone, was found in both the ring gland and the CNS of Neobellieria. Neb-AKH-GK had previously been characterised as one of the most abundant peptide peaks in Neobellieria adult corpora cardiaca [14]. Here, we show that it is present in the larval nervous system as well. FMRFamide-related peptides FMRFamide-related peptides (FaRPs) are a diverse and well-studied group of neuropeptides in arthropods ([27] for a review). The presence of FMRFamide-like

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Fig. 2. Collision induced dissociation (CID) spectra of TPSQDFMRFamide or Neb-FMRFamide-2 (upper panel) and SPPSQDFMRFamide or Neb-FMRFamide-3. In the tables, a-type, b-type, y-type, and z-type fragment ions are indicated. Theoretical fragment ion masses found in the spectrum are indicated in bold. Mass differences between expected and observed fragment ions are indicated by d.

immunoreactivity in the ventral ganglion of Neobellieria throughout metamorphosis was demonstrated [16]. We found 7 FMRFamides in the larval CNS of Neobellieria (Table 1, Fig. 2), which we named Neb-FMRFamide-2 to -8, since Neb-FMRFamide-1 had already been identified [8]. The exact N-terminal amino acid sequence of Neb-FMRFamide-7 remained uncertain, because two putative sequences, EEDFMRFamide and GGKMFMRFamide, fitted the fragmentation spectrum. Interestingly, the N-terminal sequences of the FMRFamides within the group of blowflies are remarkably conserved and differ substantially from the ones in Drosophila species. For instance, Neb-

FMRFamide-2 or TPSQDFMRFamide (Fig. 3) is identical to CalliFMRFamide-2 [28]. Furthermore, Neb-FMRFamides-1 to -5 share a C-terminal QDFMRFamide sequence. The FMRFamide genes of Calliphora vomitaria and Lucilia cuprina encoded also 6, respectively, seven different -QDFMRFamides [27], while the FMRFamide genes of D. melanogaster and virilis encoded only two different -QDFMRFamides [29]. Also Neb-FMRFamide-8 is identical to a predicted Lucilia FMRFamide, whereas no obvious homologue can be found in the Drosophila species. Finally, Neb-FIRFamide-1 is identical to CalliFIRFamide, and in Lucilia only a T residue replaces the Nterminal A residue.

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Fig. 3. Collision induced dissociation (CID) spectra of FDFHTVamide or Neb-TVamide (upper panel) and SV /W K /Q FK /Q PRL /I amide or Neb-pyrokinin-2. In the tables, a-type, b-type, y-type, and z-type fragment ions are indicated. Theoretical fragment ion masses found in the spectrum are indicated in bold. Mass differences between expected and observed fragment ions are indicated by d.

Myosuppressins are considered as members of the FaRP-family, within dipteran species they are remarkably conserved. Neb-MS is identical to the isolated Dromyosuppressin [30] and the predicted Anopheles MS [31]. Neb-myosuppressin is a myoinhibiting neuropeptide formerly isolated from head extracts of 42,000 flesh flies [11]. We identified Neb-MS by means of tandem MS (Fig. 4) in a larval CNS fraction (40 equivalents), as well as in the ring gland (four equivalents). Periviscerokinins and pyrokinins Members of the pyrokinin neuropeptide family appear to be widespread in several insect orders and even in crustaceans [32–37]. Pyrokinins were found to serve several diverse functions, including induction of sex

pheromone production in females of various moth species [34,38], stimulation of visceral muscle contractions [32], induction of cuticle colouration in caterpillars [35], induction of embryonic diapause in silkworm moth eggs [33], accelerating pupariation [39], and influencing pupal development [40]. Members of the periviscerokinin/ CAP2b family might play a role in water balance as well as in the regulation of muscle activity ([41] for a summary). Recently, the putative capa-gene products Neb-PVK1, Neb-PVK-2, and a Neb-pyrokinin (Neb-PK-1) were identified by mass spectrometry in perisympathetic organ (PSO) preparations from adult flesh flies [15]. We found these three capa-gene products also in the HPCLfractions of the larval Neobellieria CNS along with a second Neb-pyrokinin, which we named Neb-PK-2

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Fig. 4. Collision induced dissociation (CID) spectra of AYRKPPFNGSLFamide or Neb-LFamide (upper panel) and TDVDHVFLRFamide or Neb-myosuppressin. In the tables, a-type, b-type, y-type, and z-type fragment ions are indicated. Theoretical fragment ion masses found in the spectrum are indicated in bold. Mass differences between expected and observed fragment ions are indicated by d.

(Table 1). Contrary to the capa-gene products, this NebPK-2 was also present in the ring gland. Despite extensive analysis by mass spectrometry, the amino acid sequence of Neb-PK-2 remained uncertain, because several sequences fitted the fragmentation spectrum: SV /W K /Q FK /Q PRL /I amide (Fig. 3). Nevertheless, this Neb-PK-2 can be the homologue of the only other pyrokinin so far found in Drosophila: Drm-PK-2 or SVPFKPRLamide. Drm-PK-2 was one of the 28 most abundant neuropeptides in the larval CNS identified by peptidomics [3]. In addition, HPLC fraction 14 of the Neobellieria CNS contained an ion peak (m=z: 764.43) corresponding to a novel peptide, FDFHTVamide (Fig. 3), which we designated as Neb-TVamide. This peptide displays only a moderate sequence homology (FXFXXVamide) to Neb-PVK-2.

Conserved peptides Like proctolin, corazonin, and LFamide are remarkably conserved throughout arthropods. [Arg7 ]-corazonin was originally identified from the corpora cardiaca of the cockroach, Periplaneta americana, as a potent cardio-accelerating peptide [42]. [His7 ]-corazonin was identified from the corpora cardiaca of L. migratoria and Schistocerca gregaria as a dark-colour-inducing neuropeptide [43]. Recently, corazonin was even found in pericardial organs of the Jonah crab C. borealis [44]. We found [Arg7 ]-corazonin in the larval CNS fraction 29 as well as in the ring gland. The function of [Arg7 ]-corazonin in dipteran species is not yet known. Neb-LFamide was originally identified in the adult grey flesh fly and was shown to stimulate the contractions of the locust oviduct [10]. We detected this peptide

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References

Fig. 5. MALDI-TOF mass spectra from a ring gland extract, monoisotopic [M + Hþ ] are given. The (partial) sequences of Neb-PK-2 and Neb-myosuppressin were confirmed using ESI-Qqoa-TOF MS.

by tandem MS in HPLC fraction 31 using only an equivalent of 40 larval CNSs of N. bullata (Fig. 4). The impact of the advances in mass spectrometry is obvious as purification of Neb-LFamide originally required the sacrifice of 350,000 adult grey flesh flies and seven successive chromatographic separation steps [10]. In silico data mining revealed the Drosophila homologue DrmIFa, where only the penultimate L residue is replaced by an I residue [45], and the Anopheles homologue GYRKPPFNGSIFamide [31]. The same sequence as AgamIFa was recently found in diverse crustaceans: in the brain-thoracic ganglion of C. borealis [20], in the brains of the crayfish Procambarus clarkii [46], and in the eyestalk of the giant tiger prawn Penaeus monodon as FLP-7 (FMRFamide-like peptide 7) [47]. Although this peptide is widely present in the CNS of arthropods, little is known about its physiological role. In conclusion, we found 18 peptides, 11 of which are novel Neobellieria neuropeptides, in the CNS of wandering stage larvae by a peptidomics approach. Not all sequences were unequivocally determined, due to the lack of a genomic database against which mass spectrometric fragmentation spectra could be matched. Nevertheless, we substantially increased our knowledge of the peptidome of N. bullata, which is an important physiological model and a widely distributed pest fly as well. Acknowledgments P. Verleyen and J. Huybrechts are research assistants, Dr. E. Clynen and G. Baggerman are postdoctoral researchers of the Fund for Scientific Research-Flanders (Belgium) (F.W.O.-Vlaanderen). Research was supported by the FWO Grants G.0175.02 and G.O146.03. We thank R. Jonckers for the ample supply of the flesh fly.

[1] M.D. Adams et al., The genome sequence of Drosophila melanogaster, Science 287 (2000) 2185–2195. [2] R.A. Holt et al., The genome sequence of the malaria mosquito Anopheles gambiae, Science 298 (2002) 129–149. [3] G. Baggerman, A. Cerstiaens, A. De Loof, L. Schoofs, Peptidomics of the larval Drosophila melanogaster central nervous system, J. Biol. Chem. 277 (2002) 40368–40374. [4] P. Verleyen, G. Baggerman, U. Wiehart, E. Schoeters, A. Van Lommel, A. De Loof, L. Schoofs, Expression of a novel neuropeptide, NVGTLARDFQLPIPNamide, in the larval and adult brain of Drosophila melanogaster, J. Neurochem. 88 (2004) 311–319. [5] G. Cazzamali, N. Saxild, C. Grimmelikhuijzen, Molecular cloning and functional expression of a Drosophila corazonin receptor, Biochem. Biophys. Res. Commun. 298 (2002) 31–36. [6] K. Egerod, E. Reynisson, F. Hauser, M. Williamson, G. Cazzamali, C. Grimmelikhuijzen, Molecular identification of the first insect proctolin receptor, Biochem. Biophys. Res. Commun. 306 (2003) 437–442. [7] K. Egerod, E. Reynisson, F. Hauser, G. Cazzamali, M. Williamson, C. Grimmelikhuijzen, Molecular cloning and functional expression of the first two specific insect myosuppressin receptors, Proc. Natl. Acad. Sci. USA 100 (2003) 9808–9813. [8] T. Meeusen, I. Mertens, E. Clynen, G. Baggerman, R. Nichols, R. Nachman, R. Huybrechts, A. De Loof, L. Schoofs, Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor, Proc. Natl. Acad. Sci. USA 99 (2002) 15363–15368. [9] C. Rosenkilde, G. Cazzamali, M. Williamson, F. Hauser, L. Sondergaard, R. DeLotto, C. Grimmelikhuijzen, Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2, Biochem. Biophys. Res. Commun. 309 (2003) 485–494. [10] I. Janssen, L. Schoofs, K. Spittaels, H. Neven, J. Vanden Broeck, B. Devreese, J. Van Beeumen, J. Shabanowitz, D. Hunt, A. De Loof, Isolation of NEB-LFamide, a novel myotropic neuropeptide from the grey fleshfly, Mol. Cell. Endocrinol. 117 (1996) 157–165. [11] A. Fonagy, L. Schoofs, P. Proost, J. Van Damme, H. Bueds, A. De Loof, Isolation, primary structure and synthesis of neomyosuppressin, a myoinhibiting neuropeptide from the grey fleshfly, Neobellieria bullata, Comp. Biochem. Physiol. C 102 (1992) 239– 245. [12] A. Fonagy, L. Schoofs, P. Proost, J. Van Damme, A. De Loof, Isolation and primary structure of two sulfakinin-like peptides from the fleshfly, Neobellieria bullata, Comp. Biochem. Physiol. C 103 (1992) 135–142. [13] R. Nachman, G. Coast, S. Tichy, D. Russell, J. Miller, R. Predel, Occurrence of insect kinins in the flesh fly, stable fly and horn flymass spectrometric identification from single nerves and diuretic activity, Peptides 23 (2002) 1885–1894. [14] G. Baggerman, A. Cerstiaens, L. Vanden Bosch, J. Huybrechts, E. Clynen, P. Verleyen, T. Vercammen, L. Schoofs, M. Begum, A. De Loof, Search for candidate gonadotropins in extracts of the corpus cardiacum of the fleshfly, Neobellieria bullata, Invertebr. Reprod. Dev. 41 (2002) 119–125. [15] R. Predel, W. Russell, S. Tichy, D. Russell, R. Nachman, Mass spectrometric analysis of putative capa-gene products in Musca domestica and Neobellieria bullata, Peptides 24 (2003) 1487– 1491. [16] P. Sivasubramanian, FMRFamide-like immunoreactivity in the ventral ganglion of the fly Sarcophaga bullata: metamorphic changes, Comp. Biochem. Physiol. C 99 (1991) 507–512. [17] P. Sivasubramanian, Localization of leucokinin-like immunoreactive neurons and their metamorphic changes in the ventral

770

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

P. Verleyen et al. / Biochemical and Biophysical Research Communications 316 (2004) 763–770 ganglion of the fly, Sarcophaga bullata, Comp. Biochem. Physiol. A 109 (1994) 151–155. P. Sivasubramanian, P. Sood, Allatostatin-like immunoreactivity in the optic lobe of the fly Sarcophaga bullata, Cell. Mol. Biol. 49 (2003) 641–644. E. Clynen, G. Baggerman, D. Veelaert, A. Cerstiaens, D. Van der Horst, L. Harthoorn, R. Derua, E. Waelkens, A. De Loof, L. Schoofs, Peptidomics of the pars intercerebralis–corpus cardiacum complex of the migratory locust, Locusta migratoria, Eur. J. Biochem. 268 (2001) 1929–1939. J. Huybrechts, M. Nusbaum, L. Vanden Bosch, G. Baggerman, A. De Loof, L. Schoofs, Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab, Cancer borealis, Biochem. Biophys. Res. Commun. 308 (2003) 535–544. R. Huybrechts, A. De Loof, Induction of vitellogenin synthesis in male Sarcophaga bullata by ecdysterone, J. Insect Physiol. 23 (1977) 1359–1362. E. Vierstraete, A. Cerstiaens, G. Baggerman, G. Van den Bergh, A. De Loof, L. Schoofs, Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins, Biochem. Biophys. Res. Commun. 304 (2003) 831–838. A. Starratt, B. Brown, Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects, Life Sci. 17 (1975) 1253– 1256. D. Nassel, Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones, Prog. Neurobiol. 68 (2002) 1–84. E. Johnson, S. Garczynski, D. Park, J. Crim, D. Nassel, P. Taghert, Identification and characterization of a G proteincoupled receptor for the neuropeptide proctolin in Drosophila melanogaster, Proc. Natl. Acad. Sci. USA 100 (2003) 6198–6203. C. Taylor, A. Winther, R. Siviter, A. Shirras, R. Isaac, D. Nassel, Identification of a proctolin preprohormone gene (Proct) of Drosophila melanogaster: expression and predicted prohormone processing, J. Neurobiol. 58 (2004) 379–391. I. Orchard, A. Lange, W. Bendena, FMRFamide-related peptides: a multifunctional family of structurally related neuropeptides in insects, Adv. Insect Physiol. 28 (2001) 267–329. H. Duve, A. Johnsen, J. Sewell, A. Scott, I. Orchard, J. Rehfeld, A. Thorpe, Isolation, structure, and activity of –Phe–Met–Arg– Phe–NH2 neuropeptides (designated calliFMRFamides) from the blowfly Calliphora vomitoria, Proc. Natl. Acad. Sci. USA 89 (1992) 2326–2330. P. Taghert, FMRFamide neuropeptides and neuropeptide-associated enzymes in Drosophila, Microsc. Res. Tech. 45 (1999) 80– 95. R. Nichols, Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide-containing neural peptides, J. Mol. Neurosci. 3 (1992) 213–218. M. Riehle, S. Garczynski, J. Crim, C. Hill, M. Brown, Neuropeptides and peptide hormones in Anopheles gambiae, Science 298 (2002) 172–175. G. Holman, B. Cook, R. Nachman, Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae, Comp. Biochem. Physiol. C 85 (1986) 219–224. K. Imai, T. Konno, Y. Nakazawa, T. Komiya, M. Isobe, K. Koga, T. Goto, T. Yaginuma, K. Sakakibara, K. Hasegawa, O.

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45] [46]

[47]

Yamashita, Isolation and structure of a diapause hormone of the silkworm, Bombyx mori, Proc. Jpn. Acad. (1991) 98–107. A. Kitamura, H. Nagasawa, H. Kataoka, T. Inoue, S. Matsumoto, T. Ando, A. Suzuki, Amino acid sequence of pheromone-biosynthesis-activating neuropeptide (PBAN) of the silkworm, Bombyx mori, Biochem. Biophys. Res. Commun. 163 (1989) 520–526. S. Matsumoto, A. Kitamura, H. Nagasawa, H. Kataoka, C. Orikasa, T. Mitsui, A. Suzuki, Functional diversity of a neurohormone produced by the suboesophageal ganglion: molecular identity of melanization and reddish colouration hormone and pheromone biosynthesis activating neuropeptide, J. Insect Physiol. 36 (1990) 427–432. L. Schoofs, D. Veelaert, J. Vanden Broeck, A. De Loof, Peptides in the locusts, Locusta migratoria and Schistocerca gregaria, Peptides 18 (1997) 145–156. P. Torfs, J. Nieto, A. Cerstiaens, D. Boon, G. Baggerman, C. Poulos, E. Waelkens, R. Derua, J. Calderon, A. De Loof, L. Schoofs, Pyrokinin neuropeptides in a crustacean. Isolation and identification in the white shrimp Penaeus vannamei, Eur. J. Biochem. 268 (2001) 149–154. A. Fonagy, L. Schoofs, S. Matsumoto, A. De Loof, T. Mitsui, Functional cross-reactivities of some locustamyotropins and Bombyx pheromone biosynthesis activating neuropeptide, J. Insect Physiol. 38 (1992) 651–657. J. Zdarek, R. Nachman, T. Hayes, Insect neuropeptides of the pyrokinin/PBAN family accelerate pupariation in the fleshfly (Sarcophaga bullata) larvae, Ann. N. Y. Acad. Sci. 814 (1997) 67– 72. J. Sun, T. Zhang, Q. Zhang, W. Xu, Effect of the brain and suboesophageal ganglion on pupal development in Helicoverpa armigera through regulation of FXPRLamide neuropeptides, Regul. Pept. 116 (2003) 163–171. C. Wegener, Z. Herbert, M. Eckert, R. Predel, The periviscerokinin (PVK) peptide family in insects: evidence for the inclusion of CAP(2b) as a PVK family member, Peptides 23 (2002) 605–611. J. Veenstra, Isolation and structure of corazonin, a cardioactive peptide from the American cockroach, FEBS Lett. 250 (1989) 231–234. A. Tawfik, S. Tanaka, A. De Loof, L. Schoofs, G. Baggerman, E. Waelkens, R. Derua, Y. Milner, Y. Yerushalmi, M. Pener, Identification of the gregarization-associated dark-pigmentotropin in locusts through an albino mutant, Proc. Natl. Acad. Sci. USA 96 (1999) 7083–7087. L. Li, W. Kelley, C. Billimoria, A. Christie, S. Pulver, J. Sweedler, E. Marder, Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis, J. Neurochem. 87 (2003) 642–656. J. Vanden Broeck, Neuropeptides and their precursors in the fruitfly, Drosophila melanogaster, Peptides 22 (2001) 241–254. A. Yasuda, Y. Yasuda-Kamatani, M. Nozaki, T. Nakajima, Identification of GYRKPPFNGSIFamide (crustacean-SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis, Gen. Comp. Endocrinol. 135 (2004) 391–400. P. Sithigorngul, J. Pupuem, C. Krungkasem, S. Longyant, P. Chaivisuthangkura, W. Sithigorngul, A. Petsom, Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon, Comp. Biochem. Physiol. B 131 (2002) 325–337.