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Department of Animal Physiology and Neurobiology, Zoological Institute K U Leuven, Naamsestraat 59, PO Box 02465, B-3000 Leuven, Belgium
(Correspondence should be addressed to J Vanden Broeck Email: jozef.vandenbroeck{at}bio.kuleuven.be)
* *(L Badisco and I Claeys contributed equally to this work) 
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Locusts exhibit an extreme form of phenotypic plasticity: they can develop into two distinct ‘phases’, the solitarious and gregarious phase (Pener & Yerushalmi 1998). Solitarious locusts tend to avoid each other whereas gregarious animals are likely to aggregate. Combined with a high reproductive capacity, this aggregation behavior can lead to the formation of huge locust swarms consisting of billions of individuals. Solitarious and gregarious locusts not only differ in behavior, but also display remarkable morphological and physiological differences (Uvarov 1966, Pener & Yerushalmi 1998). In adults, the phase shift leads to important changes in reproductive physiology. The complete switch from one phase to the other (i.e. phase transition) usually requires several generations and is reversible. In addition to the insect developmental hormones, juvenile hormone and 20-hydroxyecdysone, brain-derived peptides, such as corazonin, adipokinetic hormone and several ‘parsins’, were already suggested to be linked to this process (Ayali et al. 1996a,b, Tawfik et al. 1999). Moreover, a phase-dependent regulation of locust neuroparsin (NPs) transcript levels was reported recently (Claeys et al. 2005). The ‘parsins’ were initially discovered as small neurosecretory proteins that are present in the pars intercerebralis–CC complex of the locust brain (Girardie et al. 1989, 1998, Lagueux et al. 1990, Hetru et al. 1991). Although NPs were first identified in locusts, genome and expressed sequence tags data have revealed the presence of various members of the NPs family in other arthropod species (Claeys et al. 2003). Furthermore, these small Cys-rich proteins display sequence similarity with the conserved N-terminal region of insulin-like growth factor binding proteins (IGFBPs), which possesses the hormone binding capacity (Claeys et al. 2003, Badisco et al. 2007). An IGFBP-like peptide was also identified in another invertebrate, namely the mollusk, Haliotis laevigata (Weiss et al. 2001). This molluskan IGFBP-like peptide, termed ‘perlustrin’, was indeed shown to interact in vitro with vertebrate ILPs, such as IGF and insulin. In adult female mosquito ovaries, a NPs-like factor, the ‘ovary ecdysteroidogenic hormone’ (OEH), displays ecdysteroidogenic activity (Brown et al. 1998) in a similar way to (vertebrate) insulin (Riehle & Brown 1999). Altogether, these previous studies suggest the possible existence of a functional relationship between both IRP and NPs-like proteins in insects. Therefore, based on our interest in studying the role of parsins in desert locust reproduction and phase transition, we were eager to identify the IRP of Schistocerca gregaria, to clone the corresponding cDNA and to analyze possible tissue-, gender- and phase-dependent differences in its transcript levels. In addition, we tried to provide experimental evidence for the hypothesis that locust NPs are capable of interacting with this endogenous locust IRP.
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Materials and methods |
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Gregarious desert locusts, S. gregaria (Forskål), were reared under crowded conditions according to the method previously described by Vanden Broeck et al. (1998). Breeding of solitarious desert locusts was performed under isolated conditions as described by Hoste et al. (2002). Temperature, photoperiod and food supply were similar for both phases.
Purification of Scg-IRP
Preparation of CC extract
A total of 1800 locust CC were micro-dissected, rinsed in a Ringer solution (1L: 8.766 g NaCl; 0.188 g CaCl2; 0.746 g KCl; 0.407 g MgCl2; 0.336 g NaHCO3; 30.807 g sucrose; 1.892 g trehalose; pH 7.2) and collected in chilled acidified ethanol (75% EtOH, 0.2 M HCl). This solution was ultrasonically homogenized (Sanyo MSE Soniprep 150) and subsequently centrifuged at 10 000 g for 30 min (Sorvall, Beckmann, Germany). The remaining pellet was extracted once again with acidified ethanol. Both supernatants were pooled and the organic solvent was evaporated (Büchi Rotavapor, Büchi Laboratory Equipment, Flawil, Switzerland). The remaining aqueous solution was delipidated by ethylacetate and n-hexane extractions. Remnants of organic solvents were removed in the rotavapor and the resulting extract was used for HPLC analysis.
High performance liquid chromatography
The first separation step was performed on a Waters Delta 600 HPLC. The chromatography was carried out on a preparative Deltapack C4 column (25 mmx100 mm; particle size 15 µm, 100 Å) at ambient temperature and flow rate of 12 ml/min. The prepared sample was then transferred to the injector and immediately after injection a linear gradient, from 0% to 90% CH3CN containing 0.1% trifluoroacetic (TFA), was initiated. The eluting peptides were detected with a variable wavelength u.v. detector, set at 214 nm (Waters 2487). Fractions of 12 ml were collected every minute, from 0 to 90 min. Aliquots (1/60 volume) of these fractions were tested in a dot blot assay, using an antibody against Lom-IRP (Riehle et al. 2006). The most intensely stained fraction was evaporated to remove CH3CN and subsequently run on a Gilson HPLC system. The separation of the compounds in this second run was carried out on a Waters semi-preparative Spherisorb C1 column (10 mmx250 mm, particle size 10 µm) at ambient temperature and at a flow rate of 2 ml/min. Immediately after injection, a linear gradient was started from 2% to 50% CH3CN containing 0.1% TFA. The eluting peptides were detected with a variable wavelength u.v. detector, which was set at 214 nm (Waters 486 Tunable Absorbance Detector). Fractions of 2 ml were collected every minute, from 0 to 60 min. Small aliquots (1/60 volume) were again sacrificed in a dot blot assay.
Dot blot assay
Aliquots of HPLC fractions were evaporated in a vacuum centrifuge and subsequently dissolved in 5 µl 10% CH3CN/0.1% TFA. Two microliters of each sample was spotted onto a nitrocellulose membrane (Hybond-C, Amersham), which was then baked for 30 min at 120 °C. Membranes were blocked with 3% skimmed milk in 50 mM Tris-buffered saline and incubated overnight at 4 °C with 1:500 diluted primary antiserum. The primary antibody, prepared in rabbit and directed against the A-chain of Lom-IRP, was a kind gift of Prof. M Brown (Department of Entomology, University of Georgia, Athens, GA, USA; Riehle et al. 2006). After rinsing, blots were incubated with a goat anti-rabbit horseradish peroxidase conjugated antibody (Dako, Carpinteria, CA, USA) for 45 min, rinsed and developed with 3,3'-diaminobenzidine (DAB) as substrate (Sigma–Aldrich).
Mass analysis and amino acid sequencing
Immunopositive fractions were subsequently analyzed by mass spectrometry (MALDI-TOF; Reflex IV, Brüker daltonics GmbH, Bremen, Germany). Aliquots of samples were loaded on a multi-sample target using 
-cyano-4-hydroxycinnamic acid as matrix and measured in linear mode. A fraction containing a potential IRP was further sequenced by Edman degradation. Therefore, the fraction was dried in a vacuum centrifuge and reconstituted in 10 µl acetonitrile/water/TFA (50:49.9:0.1 v/v/v) solution. N-terminal amino acid sequencing was carried out on a Procise 491 micro-sequencer (Applied Biosystems). The amino acids were detected by a 785-A Programmable Absorbance Detector (Applied Biosystems). Reagents required for the Edman degradation and solvents required for gradient elution were obtained from Applied Biosystems. Amino acids were identified by comparison with a standard mixture (Procise Software, Applied Biosystems, Foster City, CA, USA).
Immunocytochemistry
Locust brains were micro-dissected, rinsed in Ringer solution, and then transferred to Bouin Hollande's (10%) sublimate fixative (18–24 h). Fixed tissues were rinsed with distilled water for 12 h, dehydrated in an ethanol series, cleared overnight in histosol/paraplast (50:50 v/v) and embedded in paraplast. Alternating sections of 4 µm were made with a LKB Historange glass microtome (LKB, Stockholm, Sweden). Sections were rehydrated and processed according to the peroxidase anti-peroxidase immunocytochemistry method (Vandesande & Dierickx 1976) using DAB as chromogenic substrate. The primary rabbit antibody, raised against the A-chain of Lom-IRP (Riehle et al. 2006), was applied in a dilution of 1/500.
Preparation of RNA and cDNA
Desert locust tissues were micro- dissected under a binocular microscope and immediately collected in RNAlater solution (Ambion) to prevent degradation. Until further processing, pooled tissue samples (each sample was derived from ten individuals) were stored at –20 °C. Samples were added to reaction tubes containing Green Beads and homogenized in the MagNA Lyser instrument (Roche). Subsequently, total RNA was extracted from the resulting homogenates utilizing the RNeasy Lipid Tissue Mini Kit (Qiagen). In combination with this extraction procedure, a DNase treatment (RNase-free DNase set, Qiagen) was performed to eliminate potential genomic DNA contamination. After spectrophotometric quantification and quality control with the Agilent 2100 Bioanalyser (Agilent Technologies), 1 µg of the resulting total RNA was reverse transcribed (Superscript II, Invitrogen) utilizing random hexamers according to the company's protocol. Afterwards, the resulting cDNA was diluted tenfold.
Cloning of the Scg-IRP cDNA
Partial cDNA cloning by PCR
The PCR primer sets (I and II) were initially based on the amino acid sequence of the A- and B-chains of Scg-IRP (cf. Edman degradation) and on the nucleotide sequence of Lom-IRP. These primers had the following sequences:
5'-CGGCGAGAAGCTCTCCAA-3' (I) 
5'-CCTTCTTGAACATGGTGTTGTAGTTG-3' (I) 5'-GACGAGTGCTGCCGCAAGA-3' (II) 5'-TAGCGGCGGCCGCAGTAG-3' (II). Since sequence information of the resulting PCR fragments revealed that the coding sequences of Scg-IRP and Lom-IRP were nearly identical, a new set of primers spanning the open reading frame (ORF) was designed based on the Lom-IRP coding sequence:
5'-ATGTGGAAGCTGTGCCTCCGACTGCTCG-3' 5'-GGCCGCAGTAGGTCTGCAGCTCGCTGAT-3'. The PCR was performed with Pwo SuperYield DNA Polymerase (Roche), because the amplification efficiency appeared to be low when Taq polymerase was used, probably due to the presence of GC-rich regions in the Scg-IRP cDNA. The total reaction volume was 50 µl, including 5 µl cDNA template, 5 µl 10x PCR buffer (Roche), 1 µl dNTP mix (2.5 mM for each dNTP), 10 µl GC-rich resolution solution (Roche), 5 µl each primer (10 µM) and 0.5 µl Pwo SuperYield DNA polymerase (5 U/µl, Roche). Hot-start PCR was performed in a Thermocycler (Biometra, Göttingen, Germany). After an initial incubation at 95 °C for 2 min, thermal cycling (45 cycles) consisted of a denaturation step at 95 °C, an annealing step at 70 °C and an extension step at 68 °C, for 1 min each. A final extension step was applied at 72 °C for 7 min.
Rapid amplification of cDNA Ends (RAcE)
Based on the sequence of the ORF fragment, the following primers were designed for RAcE.
Primer for 5'-RAcE: 5'-GTTGGAGAGCTTCTCGCCGCA-3'
Primer for 3'-RAcE: 5'-GGCTTCCCAAGATGTGTCGGACGCGG-3'.
The RAcE reactions were performed with the BD SMART RAcE cDNA amplification kit (BD Biosciences, Clontech, San Jose, CA, USA) according to the manufacturer's protocol. However, due to amplification difficulties, Pwo SuperYield DNA polymerase had to be employed for the 3'-RAcE reaction, instead of Taq polymerase. The following temperature profile was applied: 5 cycles with a denaturation step at 94 °C for 30 s and annealing/extension at 72 °C for 3 min, 5 cycles with denaturation at 94 °C for 30 s, annealing at 70 °C for 30 s and extension at 72 °C for 3 min, and 40 cycles with denaturation at 94 °C for 30 s, annealing at 68 °C for 30 s and extension at 72 °C for 3 min.
Analysis of PCR and RAcE fragments
Amplification products were analyzed by horizontal agarose gel electrophoresis and purified using the GenElute gel extraction kit (Sigma–Aldrich). The DNA fragments were subcloned into the pCRII vector via the TOPO TA Cloning Kit (Invitrogen). The DNA sequences were determined using the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) following the protocols outlined in the ABI PRISM BigDye Terminator Ready Reaction Cycle Sequencing Kit (Applied Biosystems).
Quantitative real-time RT-PCR
The RT-PCRs were performed in 25 µl volume, containing 5 µl diluted cDNA sample, according to the Power SYBR Green PCR Master Mix protocol (Applied Biosystems). The final concentration of the primers was 300 nM. In order to compensate for possible variations due to pipetting errors and differences in reverse transcriptase efficiency, a S. gregaria β-actin transcript was analyzed as an endogenous control. Our previous studies indicated that the levels of this mRNA remain quite constant in locust tissues, regardless of developmental or physiological conditions (Vanden Broeck et al. 1998, Janssen et al. 2001, Claeys et al. 2003). Primers for the endogenous control were described previously (Claeys et al. 2005). Those for the Scg-IRP, target sequence were designed by means of the Primer Express software package (Applied Biosystems):
5'-CCGTGGCAACTACAACACCAT-3' 5'-TCCGCGTCCGACACATCT-3'. The reactions were run in duplicate on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) applying the following thermal cycling profile: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Data were analyzed according to the comparative CT method by means of the ABI PRISM 7000 SDS software (Applied Biosystems, version 1.2.3). The specificity of the PCRs was double-checked. Analysis of the dissociation curves of Scg-IRP and β-actin amplification products revealed a single melting peak. In addition, PCR products were analyzed via agarose gel electrophoresis, showing the presence of a single band of the expected size for each transcript. Furthermore, sequencing of the PCR products ultimately confirmed the identity of the amplified DNA. For each sample, the relative amount of transcript was normalized to the endogenous control and transcript levels were calculated relative to a calibrator sample (day 4 female brains). All experiments were repeated three times with independent samples. Statistical analysis was performed by means of Statistica 7.1 (StatSoft, Tulsa, OK, USA) and consisted of the Mann–Whitney U test for comparing two independent groups. A level of P<0.05 was considered significant.
Binding Of Scg-NP4
Vector for Scg-NP4 expression
The Drosophila inducible/secreted expression system (Invitrogen) results in the biosynthesis of a recombinant gene product in Drosophila Schneider 2 (S2) cells. The vector pMT/BiP/V5-His codes for an N-terminal, secretory signal peptide, which directs the product towards the culture medium, as well as a C-terminal peptide containing a V5-epitope for antibody detection and a polyhistidine (6x His) tag for binding onto a nickel column and subsequent affinity purification of the protein of interest. Furthermore, the expression of recombinant product is controlled by a metallothionein gene promoter (pMT). Transcription of metallothionein genes is regulated in a heavy metal-dependent manner. Hence, expression of pMT-controlled genes can be induced by adding copper ions (or other heavy metal ions) to the cell culture medium.
Based on the nucleotide sequence of the Scg-NP4 precursor (Claeys et al. 2003), a set of primers was designed for selective amplification of the cDNA fragment that codes for Scg-NP4. The upstream primer contained a BglII restriction site whereas the downstream primer included a recognition site for XhoI. Since both sites are also present in the multiple cloning site of the pMT/BiP/V5-His vector, this allowed for directional insertion of the fragment. Digestion of pMT/BiP/V5-His and the amplified fragment was performed with BglII and XhoI (Roche). After ligation (Rapid DNA Ligation Kit, Roche) and transformation in Escherichia coli DH5 cells (Invitrogen), the expression construct was verified by DNA sequencing. Subsequently, recombinant colonies were subjected to an endotoxin-free Maxiprep (Qiagen) procedure.
Biosynthesis and purification of Scg-NP4
The Drosophila S2 cells were cultured at 23 °C in Schneider's Drosophila medium (Serva Electrophoresis GmbH, Heidelberg, Germany) supplemented with 5.45 mM CaCl2, 4.44 mM NaHCO3, 50 U/ml penicillin, and 50 µg/ml streptomycin (Invitrogen) and 10% heat-inactivated fetal calf serum (Invitrogen). Transfections were carried out in serum-free Schneider's Drosophila medium with 6 µl Cellfectin (Invitrogen) and 1 µg DNA per 106 cells. Co-transfection was performed with a pCoHygro selection plasmid (carrying a hygromycin B resistance gene) in order to obtain a stable cell population. For peptide production, hygromycin B selected cells were grown in Drosophila serum-free medium (Invitrogen). After the cells had reached the exponential phase of their growth curve (2–4x106 cells/ml), recombinant NPs expression was induced (48 h) by adding CuSO4 to a final concentration of 500 µM. Recombinant Scg-NP4-V5-His in the culture medium (240 ml) was then concentrated by means of Centricon 80 cartridges (cut-off membrane of 5000 kDa; Amicon) to a final volume of 6 ml. A 10 ml column was filled with 3 ml His-Select Nickel Affinity Gel (Sigma–Aldrich). First, the column was washed with 6 ml H2Odest and subsequently three times with 6 ml equilibration buffer (50 mM sodium phosphate, pH 8.0; 0.3 M sodium chloride; 10 mM imidazole). Then, the column was loaded with 6 ml concentrated Scg-NP4-V5-His solution. The gel and medium were incubated for 1 h at ambient temperature, keeping the gel suspended by attaching the column to a rotating wheel. Next, the column was washed eight times with 6 ml equilibration buffer. Finally, the recombinant protein was eluted with elution buffer (50 mM sodium phosphate, pH 8.0; 0.3 M sodium chloride; 200 mM imidazole) and 10 fractions of 1 ml were collected. Fractions were analyzed by means of SDS-PAGE. The eluted fractions containing the recombinant protein were desalted on PD-10 columns (Amersham-Pharmacia Biotech) and concentrated in a vacuum centrifuge to a final volume of 1 ml. The presence, purity and integrity of the protein were assessed by SDS-PAGE (Laemmli 1970) using a precast NuPAGE Novex 4–12% Bis-Tris gel, MES buffer and the protein electrophoresis system (all Invitrogen). A protein ladder (SeeBlue Prestained standard, Invitrogen) was run in parallel with an affinity purified protein sample. Following electrophoresis, the gel was stained in Coomassie Brilliant Blue solution (overnight) and proteins were visualized after destaining in a methanol acetic acid solution. As an additional control, purified material was partially sequenced by Edman degradation.
Binding assay
The Scg-NP4-V5-His (200 µl concentrated sample) was loaded onto a Ni2+-column (1 ml volume), which was pretreated as described above. After rinsing the column three times with 2 ml equilibration buffer, 200 CC equivalents of purified Scg-IRP, dissolved in 2 ml equilibration buffer, were added to the nickel resin already containing Scg-NP4-V5-His and both were incubated overnight at ambient temperature using a rotating wheel in order to keep the resin suspended. In parallel, the same amount of Scg-IRP was loaded onto an empty nickel column (which did not contain Scg-NP4-V5-His). Both resins were rinsed eight times with 2 ml equilibration buffer. Next, peptides were eluted from the resin with 2 ml elution buffer and this was repeated seven times. Wash and elution fractions were analyzed for the presence of Scg-NP4-V5-His and/or Scg-IRP by means of a dot blot assay, in which recombinant Scg-NP4-V5-His and Scg-IRP were detected by antibodies against the V5 epitope (Invitrogen) and the A-chain of Lom-IRP respectively.
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Fractions of the first HPLC run, which eluted between 29 and 36 min, resulted in positive staining (Fig. 1A) during the dot blot assay. Next, fraction 31 that was the most positively stained fraction was further analyzed by reversed-phase HPLC. From the subsequent dot blot assay, one positively immunostained fraction was obtained, which eluted at 51 minutes corresponding to 42.5% CH3CN (Fig. 1B). Part of this fraction (1/60) was subsequently analyzed by MALDI-TOF MS, revealing an average mass of 5736 kDa, which was in accordance with the expected mass of a potential ILP.
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Edman degradation revealed the complete amino acid sequence of both the A- and B-chains of Scg-IRP, with the exception of the amino acid at position 15 of the A-chain (X). Comparison of the amino acid sequence with the only other identified IRP from Orthopteroidea, namely Lom-IRP, revealed that one amino acid was different: at position 21, the B-chain of Scg-IRP contains an isoleucine (I) instead of a leucine (L) in Lom-IRP. The A-chain appears to be identical in both locusts. Hence, the antibody against the complete A-chain of Lom-IRP can also be used for specific detection of Scg-IRP.
Immunolocalization in the brain
Alternating desert locust brain sections were incubated with rabbit polyclonal antibody raised against the A-chain of Lom-IRP and the rabbit pre-immune serum respectively. Intensely stained median neurosecretory cells were observed within the pars intercerebralis. Furthermore, immunopositive staining was also observed in the neurohemal storage part of the CC and in the connecting nerves between the pars intercerebralis and the CC, namely the nervi corporis cardiaci I (NCC-I; Fig. 2).
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Sequencing of the obtained PCR fragments revealed the amino acid at position 15 as glutamic acid (E), identical to the A-chain of Lom-IRP (Fig. 3B). Next, primers for the RAcE reactions were derived from the partial cDNA sequence obtained by PCR. The RAcE-PCR strategy resulted in the identification of two transcript sequences that differed only in their 5'-untranslated region. These cDNA sequences and the deduced amino acid sequence of the encoded pre-pro-Scg-IRP precursor are represented in Fig. 3(A). When entering the Scg-IRP precursor amino acid sequence into the SignalP 3.0 algorithm (Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark) (Bendtsen et al. 2004), the most probable cleavage site for the signal peptide is between position 22 and 23, as was also observed for Lom-IRP (Clynen et al. 2003).
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In both solitarious and gregarious locusts, the presence of Scg-IRP mRNA was detectable in virtually all tissues that were tested, i.e. nervous system, fat body, gonads, male accessory glands, flight muscles and salivary glands. The Scg-IRP transcript levels were most pronounced in nervous tissue and in fat body. Figure 4 shows the relative quantities of Scg-IRP mRNA that were determined in brain and fat body of adult locusts as a function of gender and of age (4 and 10 days after adult emergence). Significant temporal fluctuation was noticed in the fat body of both adult males and females, as well as in the brain of adult gregarious females. In the fat body of gregarious males, the Scg-IRP transcripts reached a significantly higher level (ca. threefold) at day 10, when compared with day 4. This is contrary to the situation in the fat body of solitarious males, where relative Scg-IRP mRNA quantities were lower (ca. twofold) at day 10 (Fig. 4B). As a result, significant differences in the Scg-IRP transcript levels were observed between solitarious and gregarious male fat bodies at day 10. In gregarious female brains, the Scg-IRP mRNA shows a small but significant reduction in its abundance at day 10, whereas in brains of solitarious females and both solitarious and gregarious males, this remains practically constant (Fig. 4A and C). In the fat body of gregarious females, a very pronounced increase (
20-fold) in the Scg-IRP transcript levels was noticed at day 10, when compared with day 4. In solitarious female fat bodies, a much smaller increase (ca. threefold) was observed (Fig. 4D).
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Culture medium containing concentrated Scg-NP4-V5-His was subjected to a one-step nickel ion chelate affinity chromatography. All elution fractions were tested for the presence of Scg-NP4-V5-His protein by means of SDS-PAGE. Fractions containing the eluted protein were combined, desalted and concentrated. In the concentrated sample, one single protein band of 
12 kDa was observed by SDS-PAGE (Fig. 5A). This estimated molecular mass is in line with the calculated mass of Scg-NP4-V5-His (12 404.87 kDa). In addition, amino acid sequencing indeed identified the purified material as recombinant Scg-NP4-V5-His.
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In combination with this immunological screening method, our research strategy has resulted in the purification of Scg-IRP, as well as in the determination of its amino acid sequence. The Scg-IRP possesses the typical structure of a metazoan insulin-like hormone (with the exception of vertebrate IGFs or IGFs and several Caenorhabditis elegans ILPs) that consists of A- and B-chains that are interconnected by Cys-bridges. Subsequently, the corresponding cDNA was cloned by means of a PCR- and RAcE-based strategy and sequenced (Fig. 3). The encoded hormone precursor exhibits a very similar organization as observed for most other members of the insulin superfamily. The pre-pro-Scg-IRP polypeptide indeed contains a signal peptide, which directs the hormone to the secretory pathway, as well as a pro-insulin portion with a B-, C- and A-chain. Upon removal of the signal peptide and the C-chain, the A- and B-chains are covalently coupled by means of disulfide bridges and constitute the mature peptide. However, this desert locust IRP precursor still contains another peptide sequence that is situated between the signal sequence and the B-chain. This decapeptide has previously been termed ‘IRP-copeptide’. Its existence was first demonstrated in the migratory locust, Locusta migratoria, where it may be involved in the regulation of carbohydrate metabolism (Clynen et al. 2003). The IRP co-peptide is identical in L. migratoria and S. gregaria. In contrast to the well-conserved sequences of Scg-IRP and IRP-copeptide, the C-chain is clearly less conserved between locust species (Fig. 3B). This observation is not unexpected, since, for all members of the insulin superfamily, the B- and A-chains are noticeably more conserved than the C-chain (Claeys et al. 2002). Multiple alignment of the newly identified Scg-IRP sequence with other known insect IRPs (Fig. 6) show that the cystein pattern in both the A- and B-chains is extremely well conserved. In addition, a tyrosine (Y) and leucine (L) residue in the A- and B-chains respectively, appear to occur in all known insect IRPs. Therefore, the observed results concerning the primary structure of the peptide and its cDNA indicate that pre-pro-Scg-IRP shares several general characteristics with other members of the insulin superfamily, while it also possesses some properties that may be more specific for locusts. In contrast to genome-derived nucleotide sequence data obtained from lepidopteran, dipteran and hymenopteran species, only one single IRP has so far been identified in each of the orthopteran species, L. migratoria and S. gregaria. For both Lom-IRP and Scg-IRP, two transcript (cDNA) variants have now been identified, but these only differ in their 5' untranslated region (Kromer-Metzger & Lagueux 1994). Since the genomes of these species have not (yet) been sequenced, it is still unclear whether other IRP genes remain to be discovered in these locusts.
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In addition to insulin (this study), locusts possess different NPs, which are structurally related to the OEH of mosquitoes and to vertebrate IGFBP (Badisco et al. 2007). In contrast to OEH, NP (i.c. Scg-NP1) was initially discovered as a factor that prevented oocyte growth and thus delayed the gonotropic cycle (Girardie et al. 1998). At present, four NPs (Scg-NP1-4) have been identified from S. gregaria (Janssen et al. 2001, Claeys et al. 2003), and in the fat body of adult gregarious locusts the transcripts coding for Scg-NP3 and for Scg-NP4 are strongly upregulated during sexual maturation, similarly as Scg-IRP. In the present study, we demonstrate the in vitro binding of recombinant Scg-NP4 with the purified, endogenous Scg-IRP (Fig. 5). This result represents the first experimental evidence that insect NPs possess the capacity to interact with insulin-related peptides, as IGFBPs do in vertebrates and perlustrin in the mollusk, H. laevigata (Weiss et al. 2001). If such interactions would also occur in vivo, NPs may exert regulatory effects situated upstream of IRP receptor signaling. These likely include effects that modulate molecular interactions, turnover, transport and tissue targeting of the corresponding insulin-like hormone(s) (Simonet et al. 2004). However, as exemplified by IGFBPs, this does not necessarily exclude the possible existence of additional modes of action (Mohan & Baylink 2002). In the future, it will be of interest to further examine the in vivo role of Scg-IRP and to analyze whether Scg-NPs may exert an inhibiting or stimulating effect on its activities. Furthermore, more insight in the mechanisms controlling reproduction and phase transition could offer novel opportunities to improve prevention and control of locust swarms.
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Acknowledgements |
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References |
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Received in final form 21 December 2007
Accepted 10 January 2008
Made available online as an Accepted Preprint 10 January 2008
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