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Cloning and characterization of a third isoform of corazonin in the honey bee Apis mellifera
peptides 27 (2006) 493–499
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Cloning and characterization of a third isoform of corazonin in the honey bee Apis mellifera Peter Verleyen a,*, Geert Baggerman a, Inge Mertens a, Tim Vandersmissen a, Jurgen Huybrechts a, Alfons Van Lommel b, Arnold De Loof a, Liliane Schoofs a a b
Laboratory of Developmental Physiology, Genomics and Proteomics, KULeuven, Naamsestraat 59, B-3000 Leuven, Belgium Department of Morphology and Molecular Pathology, KULeuven, B-3000 Leuven, Belgium
article info
abstract
Article history:
The precursor of the insect hormone corazonin has been cloned from the honey bee Apis
Received 31 January 2005
mellifera. The precursor predicts a novel isoform of corazonin, pQTFTYSHGWTNamide,
Accepted 18 March 2005
which was confirmed by tandem mass spectrometry. Although Apis corazonin differs only
Published on line 9 January 2006
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 immunohis-
Keywords:
tochemistry of the central nervous system of the honey bee showed a pattern similar to the
Apis mellifera
ones described for other insects. Four neurons of the lateral protocerebrum project axons
Drosophila melanogaster
towards the retrocerebral complex. It is unlikely that Apis corazonin is present in all
Hymenoptera
hymenopteran species since the presence of this peptide could not be demonstrated by
Mass spectrometry
means of mass spectrometry in the retrocerebral complex of the red wood ant Formica rufa
Corazonin
and the wasp Vespula saxonica. Instead, we found masses corresponding with [Arg7]- and
Neuropeptide precursor
[His7]-corazonin respectively, suggesting that some of the corazonin isoforms originated late during evolution in different insect orders. # 2005 Elsevier Inc. All rights reserved.
1.
Introduction
Corazonin was originally purified by Veenstra from 600 corpora cardiaca (CC) of the American cockroach Periplaneta americana as the most potent insect cardioactive neuropeptide [24]. Hence the name of corazonin, from the Spanish word for heart: ‘‘corazon’’. After enzymatic deblocking of the Nterminal pyroglutamate, the sequence was determined by Edman degradation based sequencing as pQTFQYSRGWTNamide. Interestingly, a second isoform of corazonin, was identified in Schistocerca americana [25]. This [His7]-corazonin differs from the cockroach [Arg7]-corazonin only by a His/Arg substitution [25]. In addition, the presence of [His7]-corazonin was unequivocally demonstrated in the CC of three additional species: S. gregaria, Locusta migratoria [23] (Orthoptera) and Carausius morosus [16] (Phasmatodea). [Arg7]-corazonin was
also detected in Drosophila melanogaster [1], Neobellieria bullata [29] (Diptera), Nauphoeta cinerea [25] (Dictyoptera), Gryllus bimaculatus [8] (Orthoptera), Manduca sexta [25], Bombyx mori [8], Galleria mellonella [5] (Lepidoptera) and even in the crustacean species Cancer borealis [11]. Thus, the presence of [His7]- and [Arg7]-corazonin is only demonstrated for a limited number of insect species and orders. Immunohistochemical studies showed that the distribution of corazonin or corazonin-like substances in the central nervous system (CNS) is highly similar in the studied insects. In all cases, except for beetles, a group of neurons in the pars lateralis of the protocerebrum, project axons towards the ipsilateral retrocerebral neurohemal organs [2,15,17–19,27]. Despite the fact that both the sequence and distribution pattern of corazonin are highly conserved among insect species and orders, no common function of this neuropeptide
* Corresponding author. Tel.: +32 16 324260; fax: +32 16 323902. E-mail address: [email protected] (P. Verleyen). 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.03.065
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has yet been demonstrated. Surprisingly, the heart accelerating effect of corazonin was largely restricted to Pe. americana [15]. The discovery that in both S. gregaria and L. migratoria corazonin induces black patterns which are typical for the gregarious phase of these plague locusts at outbreaks [23], boosted the research on this peptide. In addition, the Drosophila corazonin receptor was cloned and functionally expressed [4,13]. Furthermore, corazonin was associated with a lot of other functions. Its release might be suppressed by high temperatures resulting in light body coloration in L. migratoria [21]. Injections of corazonin shifted the morphogenetics of isolated-reared desert locusts towards values typical for crowd-reared animals [7] and corazonin promotes tegumentary pigment migration in the crayfish Procambarus clarkii [14]. In the silkworm, B. mori corazonin reduces the spinning rate [22]. Recently, corazonin was shown to stimulate the release of pre-ecdysis and ecdysis-triggering hormones from the Inka cells of M. sexta, inducing precocious pre-ecdysis and ecdysis behaviors [10]. In the present study, we wanted to fully characterize corazonin in a hymenopteran insect. The Hymenoptera are one of the largest and economically most important insect orders. Apis mellifera in particular is a social insect and a model organism to study development, immunity and behavior.
2.
Material and methods
2.1.
Insects
2.2.
The Quickprep Micro mRNA Purification Kit (Amersham Biosciences, USA) was used to isolate the mRNA of 50 CNSs of Ap. mellifera workers. cDNA was made with the SuperScript First-Strand Synthesis System (Invitrogen, USA). The ORF of the Apis corazonin precursor was amplified by the Advantage 2 PCR Enzyme System (BD Biosciences, USA) with specific oligonucleotide PCR primers (Eurogentech, Belgium). Based on the Apis ESTdatabase [32] the forward primer was 50 -CCA GAT GGT TAA CTC TCA AAT CC-30 and the reverse primer 50 TTA ATA ATT GTT ATT GGT AGG AGC G-30 (Fig. 1, bold type). The PCR was conducted with the following parameters: first a denaturation step of 94 8C for 60 s, then 30 cycles of 95 8C for 30 s and 58 8C for 30 s, and finally an extra extension step of 68 8C for 60 s. The obtained PCR product was first cloned in the pCR4-TOPO vector using the TOPO-TA cloning kit (Invitrogen, The Netherlands). Then the vector was transformed into E. coli TOP10 (Invitrogen, The Netherlands) which were grown overnight at 37 8C in a shaking incubator. After selection of the obtained clones, the plasmid DNA was isolated with the Nucleospin Plasmid Quick Pure kit (Macherey-Nagel, Germany) and digested with EcoRI. Prior to DNA sequence analysis (310 Genetic Analyzer, Applied Biosystems, UK), the precursor DNA was amplified with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit v1.1 (Applied biosystems, UK). The PCR parameters were: 96 8C for 30 s, 50 8C for 15 s and 60 8C for 240 s (25 cycles).
2.3. Foraging workers of the honey bee Ap. mellifera carnica (Hymenoptera) were obtained from a local beekeeper. Workers of the wasp Vespula saxonica were caught daily upon emergence from an isolated nest. Workers of the red wood ant Formica rufa were caught from a large nest in Heverlee (Belgium). Albino Locusta migratoria from the Okinawa strain were reared in crowded conditions at an average temperature of 29 8C, a photoperiod of 13-h light:11-h dark and a diet of grass and oat flakes.
Cloning of the corazonin precursor in Ap. mellifera
Mass spectrometry
Forty retrocerebral complexes (corpora cardiaca and corpora allata) of Ap. mellifera were dissected for tandem MS analysis. Thirty retrocerebral complexes of Ap. mellifera, F. rufa and V. saxonica were dissected for MS analysis. Tissues were dissected and rinsed in physiological saline and extracted in 100 mL methanol/water/formic acid (FA) (90:9:1, v/v/v). The extracts were sonicated and centrifuged at 10,000 rpm for 10 min. The pellets were extracted, sonicated and centrifuged
Fig. 1 – Nucleotide sequence and the deduced amino acid sequence for the open reading frame of the cDNA encoding the Apis mellifera corazonin precursor. The sequences corresponding to the oligonucleotide primers used in the PCR amplification are in bold. The amino acid sequence reveals a signal peptide (italics), followed by corazonin (underlined), a dibasic cleavage site and the corazonin related peptide.
peptides 27 (2006) 493–499
a second time. The pooled supernatants of 40 Ap. mellifera retrocerebral complexes were filtered through a Millipore spindown filter, dried and stored at 20 8C. Capillary LC– tandem MS was performed as described [28]. In brief, 10 mL of the sample (containing an equivalent of 15 retrocerebral complexes) was loaded onto a pre-column with an isocratic flow of 2% CH3CN in water with 0.1% FA at a flow rate of 10 mL/ min. After 2 min, the pre-column was switched online with the analytical capillary column at a flow rate of 150 nL/min. Ions with m/z close to 662.23 (which is the calculated mass of Ap. mellifera corazonin (1322.57 Da) +2H+/2z) were automatically selected for fragmentation during the nano–LC tandem MS separation. The pooled supernatants of the retrocerebral complexes of Ap. mellifera, F. rufa and V. saxonica were completely evaporated and then redissolved in 25 mL water/CH3CN/trifluoroacetic acid (TFA) (97.9:2:0.1, v/v/v). A ZipTipC18 (Millipore, 15 mm) was used to concentrate and desalt the sample. Samples were eluted with 3 mL water/CH3CN/FA (69.9:30:0.1, v/v/v). One or two microlitres of the samples were transferred to a ground steel target plate and mixed with 0.5 mL a-cyano-4-hydroxycinnamic acid in acetone, air-dried and analyzed by MALDITOF MS as described [31].
2.4.
495
coli, DNA sequence analysis revealed the Apis corazonin precursor (Fig. 1). The corazonin precursor contains a signal peptide of 21 amino acid residues, followed by corazonin, a dibasic cleavage site and a corazonin associated peptide. Apis corazonin differs from [His7]-corazonin by the substitution of glutamine4 by a threonine residue (Fig. 1). Hence, Apis corazonin is the third isoform of this neuropeptide. An ion peak with a mass that matches Apis corazonin can be observed in a MALDI spectrum of retrocerebral complexes (Fig. 2A). However, the presence of this isoform in these endocrine organs of Ap. mellifera was unequivocally confirmed by means of tandem MS (Fig. 3). Note that when total brains were dissected, the ion peak corresponding with Apis corazonin
Dark color inducing bioassay
Albino Locusta migratoria from the Okinawa strain were collected after the molt to the fourth instar. Groups of 6 (control) to 12 individuals were injected on day 0 with 3 mL of rapeseed oil containing 1 nmol, 10 or 0.1 pmol of synthetic Apis corazonin. A negative control group was injected with pure rapeseed oil. As a positive control, locusts were injected with similar amounts of [His7]-corazonin. The color of the locusts was evaluated 2 days after injection and 2 days after they molted to the fifth instar. No darkening at all was observed in the pure rapeseed oil injected locusts, whereas the locusts injected with [His7]-corazonin showed a similar concentration dependent grade of black coloration as described [33].
2.5.
Whole mount immunohistochemistry
Polyclonal antisera against [His7]-corazonin were raised in New Zealand White rabbits, which were immunized with synthetic [His7]-corazonin that was conjugated to thyroglobulin by means of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and glutaraldehyde. The specificity of the antiserum was demonstrated using pre-immune antiserum. The primary antiserum was pre-absorbed with 100 mM of the synthetic peptide. The CNS of Ap. mellifera foragers was studied using the whole mount protocol as described [28].
3.
Results
3.1.
A third bioactive isoform of corazonin in Ap. mellifera
Amplification of the cDNA, derived from 50 CNSs of Apis mellifera, with the specific oligonucleotide primers resulted in a PCR product of the calculated length (slightly above 300 bp). After cloning in the pCR4-TOPO vector and transfection in E.
Fig. 2 – MALDI-TOF mass spectra from an equivalent of 10 to 20 retrocerebral complexes of the honey bee Ap. mellifera (A), the red wood ant F. rufa (B) and the wasp V. saxonica (C). Monoisotopic [M + H+] are given. An ion peak mass corresponding with the calculated mass of [Thr4, His7]-corazonin (M = 1322.57) was found in Ap. mellifera (A). An ion peak mass matching the calculated mass of [Arg7]-corazonin (M = 1368.62) was found in F. rufa (B). A small ion peak mass corresponding with the calculated mass of [His7]-corazonin (M = 1349.58) was found in V. saxonica (C).
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Fig. 3 – CID (collision induced dissociation) spectrum of pQTFTYSHGWTNamide or Apis Corazonin. a-type, b-type, y-type fragment ions are indicated. NanoLC run of 15 retrocerebral complexes of Apis mellifera.
could not be discerned (data not shown). This is most probably due to a lower concentration of corazonin in the brain. Injection of 1 nmol of synthetic Apis corazonin in albino locusts resulted in a complete darkening 2 days after the moult to the fifth instar, whereas 10 pmol caused darkening of the lowest degree in some individuals and 0.1 pmol had no effect at all (data not shown). These results are in agreement with the findings of Yerushalmi et al. who stated that the full sequence of [His7]-corazonin is required for a maximal effect [33]. Ten picomoles of the entire [His7]-corazonin sequence resulted in a complete darkening of most locusts, but an equal amount of N- or C-terminal shortened sequences had only minor effects [33].
3.2.
Corazonin immunoreactivity in Ap. mellifera
Whole mount immunohistochemistry was used to investigate the pattern of corazonin immunoreactivity in the CNS of Ap. mellifera. Pre-immune antiserum and pre-absorbed primary
antiserum did not cause any staining (not shown). Using this primary corazonin antiserum, four pairs of neurosecretory cells were observed in the lateral protocerebrum (Fig. 4). These neurons project parallel processes into the medial protocerebrum and send one bundle of axons posteriorly and ventrolaterally. This bundle turns towards the midline before leaving the brain and innervating the ipsilateral retrocerebral organs of Ap. mellifera (Fig. 5), in which corazonin was detected by means of mass spectrometry (Figs. 2A and 3). The presence of corazonin-like immunoreactivity in two groups of protocerebral lateral neurosecretory cells projecting into the ipsilateral retrocerebral neurohemal organs is consistent with a distribution pattern observed in all insect species investigated so far, except for beetles [2,15,17–19,27]. The presence of corazonin-like immunoreactive cells in the thoracic and abdominal ganglia on the other hand, shows much more variation [2,17,27]. In situ hybridization in Ga. mellonella confirmed the presence of corazonin in the lateral neurosecretory cells, whereas no corazonin precursor mRNA could be detected in other cells of the nervous system [5]. We found no obvious corazonin-like immunoreactive cells in the thoracic nor in the abdominal ganglia of Ap. mellifera.
3.3.
Fig. 4 – Detail of the corazonin immunoreactivity in the pars lateralis of a whole mount tissue preparation of the adult brain of Apis mellifera. This image was obtained by superimposing seven optical sections 1.5 mm apart. Immunoreactivity was found in four neurons with axons projecting posteriorly. Bar = 50 mm.
Is Apis corazonin the sole isoform in Hymenoptera?
A previous study has shown by means of the dark color inducing bioassay, that besides Ap. mellifera other Hymenoptera also contain a bioactive corazonin-like substance [20]. The question arises now whether or not Apis corazonin counts in particular for the dark color inducing activity of the other hymenopteran species investigated [20]. This question can be represented as: is Apis corazonin the sole isoform throughout all the species of this insect order? Therefore, we used MALDITOF mass spectrometry to scan the retrocerebral complexes of two other relatively large hymenopteran species, i.e. the wasp V. saxonica and the red wood ant F. rufa for the presence of the typical bee [Thr4, His7]-corazonin. The ion mass corresponding with this corazonin (M = 1322.57 Da) could, in contrast to Ap. mellifera (Fig. 2A), not be detected in the MALDI-TOF mass spectra of the retrocerebral complex in case of F. rufa and V. saxonica (Fig. 2B and C). However, the mass spectrum of the brain-CC extract of F. rufa shows a large peak with an ion mass corresponding to the calculated mass of [Arg7]-corazonin (M = 1368.62 Da), indicating that this isoform is present in this
peptides 27 (2006) 493–499
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to have originated late during evolution in different insect orders. The presence of corazonin-like substance in the primitive insect, the silverfish Ctenolepisma lineata [17] and even in the crustacean species, the Jonah crab Cancer borealis [11] suggests an ancient origin of this neuropeptide. In this perspective, other corazonin isoforms might be out there and beetles might not have lost corazonin but might contain a dramatically changed isoform that is inactive in the dark color inducing bioassay.
Fig. 5 – Corazonin immunoreactivity in the adult brain of Apis mellifera. Schematic drawing based on whole mount tissue preparations. Frontal view, immunoreactivity is bilaterally symmetrical, the immunoreactivity of the left hemisphere is depicted. Immunoreactivity was found in four neurons with processes into the medial protocerebrum. One bundle of axons innervates the ipsilateral corporus cardiacum (CC) via the posterior lateral tract (PLT). Esophageal opening (EF); subesophageal ganglion (SG).
ant species (Fig. 2B). The mass spectrum of the brain-CC extract of V. saxonica on the other hand, contains a small but distinct ion peak with the calculated mass of [His7]-corazonin (M = 1349.58) providing evidence that [His7]-corazonin or perhaps a fourth isoform might be present in this wasp species (Fig. 2C). Hence, our findings indicate that Apis corazonin is not present in all Hymenoptera. The presence of a particular corazonin isoform is thus not determined by the insect order. In other words, several corazonin isoforms seem
4.
Discussion
4.1. The conservation of corazonin and its precursor throughout insect species and orders Only three insect neuropeptide precursors, i.e. the adipokinetic hormone (AKH), SIFamide and corazonin precursors display a similar general structural pattern: one copy of the bioactive peptide is situated immediately after the signal peptide and separated by the common GKR processing site from the subsequent joining peptide. In addition, the Akh, Crz and IFa genes of at least D. melanogaster have in common that they are characterized by two exons separated by a small intron (www.flybase.org). Small sequence similarities between AKH and corazonin suggested an evolutionary link [24]. This hypothesis was confirmed by the fact that the Drosophila AKH and corazonin receptors are structurally related, which even implies a co-evolution of ligands and receptors [3,13]. Furthermore, the joining peptides of AKH have cysteine residues allowing the formation of dimers via cystine bridges, as was demonstrated in the locusts S. gregaria and L. migratoria [6,9]. Recently, it was found that all known
Fig. 6 – Alignment of insect corazonin precursors. Gam is Galleria mellonella, accession AY004866.1 (AAF87082.1); Bom Bombyx mori, accession AB106876.1 (BAC66443.1); Drm Drosophila melanogaster, accession NM_079626.2 (NP_524350.1; CG3302); Drp Drosophila pseudoobscura, accession AADE01000000 (AADE01000051); Apm Apis mellifera (gnljAmel_1.1jGroupUn.323); Aea Aedes aegypti (TC35312) and Ang is Anopheles gambiae, accession XM_313456.1 (ENSANGP00000020275). Sequences were retrieved by BLAST searching and processing the available genomic databases of insect species. CLUSTALW multiple alignment (www.ebi.ac.uk/clustalw) was performed. Signal peptides predicted by the SignalP program (http://www.cbs.dtu.dk/services/SignalP/[12]) are in italics. Identical amino acid residues in bold. Dibasic cleavage sites and cysteine residues are underlined.
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joining peptides of the SIFamide precursor in insect species have two cysteine residues, separated by six amino acid residues [30]. Dimerization of the relatively well conserved SIFamide precursors is thus possible as well. To investigate whether corazonin precursors contain cysteine residues, the currently available genomic databases of insects were extensively datamined and processed. The corazonin precursors of seven insect species were detected and compared (Fig. 6). The corazonin precursor of D. melanogaster was already cloned in 1994 from a genomic library [26]. A corazonin-precursor-related peptide (CPRP) of 39 amino acid residues, with an internal potential dibasic cleavage site (RR) but without cysteine residues was predicted [26]. However, an intron between amino acid residues 72 and 73 and containing a TAA stopcodon was overlooked [1]. Without this intron the Drosophila corazonin precursor does contain two cysteine residues (Fig. 6). A similar intron at about the same position was also found in the corazonin genes of Ap. mellifera, B. mori and D. pseudoobscura (data not shown). The corazonin precursors of G. mellonella and Ae. aegypti were derived from cDNA, thus it is unknown whether these corazonin genes have a similar organization. Interestingly, no such intron could be predicted in the corazonin gene of the malaria mosquito An. gambiae. Moreover, the originally annotated Anopheles corazonin precursor (Fig. 6) was replaced by a precursor containing only the four N-terminal amino acids of this precursor, which seems highly improbable. Nevertheless, all corazonin precursors display at least one cysteine residue, although the corazonin joining peptide is clearly not as conserved as it is the case for SIFamide joining peptide and AKH joining peptide. Thus, dimerization or internal cystine bridges cannot be excluded. In addition, all corazonin precursors, except for the Galleria precursor, contain at least one internal dibasic cleavage site, allowing further processing of the corazonin joining peptide (Fig. 6).
5.
Conclusion
In conclusion, we characterized a third isoform of corazonin in the honey bee Ap. mellifera, which is most likely not present in all Hymenoptera. Like [His7]- and [Arg7]-corazonin, Apis corazonin is biologically active in the locust coloration assay, although to a lesser extent. Apis corazonin is present in a group of lateral cells in the protocerebrum which innervate the retrocerebral complex. The Apis corazonin joining peptide shows minor sequence similarities with the other identified corazonin joining peptides, although the presence of cysteine residues seems conserved, as it is the case in the joining peptides of the related neuropeptides AKH and SIFamide.
Acknowledgements P. Verleyen and I. Mertens are post-docs of the KULeuven Research Council, G. Baggerman and J. Huybrechts are postdoctoral researchers of the Fund for Scientific ResearchFlanders (Belgium) (F.W.O.-Vlaanderen). Research was supported by the FWO-grants G.0175.02 and G.O146.03. We thank Mrs. Chris Heremans for the supply of Apis mellifera, Dr. Eric Schoeters and the ALRC for their assistance.
references
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Cloning and characterization of a third isoform of corazonin in the honey bee Apis mellifera Peter Verleyen a,*, Geert Baggerman a, Inge Mertens a, Tim Vandersmissen a, Jurgen Huybrechts a, Alfons Van Lommel b, Arnold De Loof a, Liliane Schoofs a a b
Laboratory of Developmental Physiology, Genomics and Proteomics, KULeuven, Naamsestraat 59, B-3000 Leuven, Belgium Department of Morphology and Molecular Pathology, KULeuven, B-3000 Leuven, Belgium
article info
abstract
Article history:
The precursor of the insect hormone corazonin has been cloned from the honey bee Apis
Received 31 January 2005
mellifera. The precursor predicts a novel isoform of corazonin, pQTFTYSHGWTNamide,
Accepted 18 March 2005
which was confirmed by tandem mass spectrometry. Although Apis corazonin differs only
Published on line 9 January 2006
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 immunohis-
Keywords:
tochemistry of the central nervous system of the honey bee showed a pattern similar to the
Apis mellifera
ones described for other insects. Four neurons of the lateral protocerebrum project axons
Drosophila melanogaster
towards the retrocerebral complex. It is unlikely that Apis corazonin is present in all
Hymenoptera
hymenopteran species since the presence of this peptide could not be demonstrated by
Mass spectrometry
means of mass spectrometry in the retrocerebral complex of the red wood ant Formica rufa
Corazonin
and the wasp Vespula saxonica. Instead, we found masses corresponding with [Arg7]- and
Neuropeptide precursor
[His7]-corazonin respectively, suggesting that some of the corazonin isoforms originated late during evolution in different insect orders. # 2005 Elsevier Inc. All rights reserved.
1.
Introduction
Corazonin was originally purified by Veenstra from 600 corpora cardiaca (CC) of the American cockroach Periplaneta americana as the most potent insect cardioactive neuropeptide [24]. Hence the name of corazonin, from the Spanish word for heart: ‘‘corazon’’. After enzymatic deblocking of the Nterminal pyroglutamate, the sequence was determined by Edman degradation based sequencing as pQTFQYSRGWTNamide. Interestingly, a second isoform of corazonin, was identified in Schistocerca americana [25]. This [His7]-corazonin differs from the cockroach [Arg7]-corazonin only by a His/Arg substitution [25]. In addition, the presence of [His7]-corazonin was unequivocally demonstrated in the CC of three additional species: S. gregaria, Locusta migratoria [23] (Orthoptera) and Carausius morosus [16] (Phasmatodea). [Arg7]-corazonin was
also detected in Drosophila melanogaster [1], Neobellieria bullata [29] (Diptera), Nauphoeta cinerea [25] (Dictyoptera), Gryllus bimaculatus [8] (Orthoptera), Manduca sexta [25], Bombyx mori [8], Galleria mellonella [5] (Lepidoptera) and even in the crustacean species Cancer borealis [11]. Thus, the presence of [His7]- and [Arg7]-corazonin is only demonstrated for a limited number of insect species and orders. Immunohistochemical studies showed that the distribution of corazonin or corazonin-like substances in the central nervous system (CNS) is highly similar in the studied insects. In all cases, except for beetles, a group of neurons in the pars lateralis of the protocerebrum, project axons towards the ipsilateral retrocerebral neurohemal organs [2,15,17–19,27]. Despite the fact that both the sequence and distribution pattern of corazonin are highly conserved among insect species and orders, no common function of this neuropeptide
* Corresponding author. Tel.: +32 16 324260; fax: +32 16 323902. E-mail address: [email protected] (P. Verleyen). 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.03.065
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has yet been demonstrated. Surprisingly, the heart accelerating effect of corazonin was largely restricted to Pe. americana [15]. The discovery that in both S. gregaria and L. migratoria corazonin induces black patterns which are typical for the gregarious phase of these plague locusts at outbreaks [23], boosted the research on this peptide. In addition, the Drosophila corazonin receptor was cloned and functionally expressed [4,13]. Furthermore, corazonin was associated with a lot of other functions. Its release might be suppressed by high temperatures resulting in light body coloration in L. migratoria [21]. Injections of corazonin shifted the morphogenetics of isolated-reared desert locusts towards values typical for crowd-reared animals [7] and corazonin promotes tegumentary pigment migration in the crayfish Procambarus clarkii [14]. In the silkworm, B. mori corazonin reduces the spinning rate [22]. Recently, corazonin was shown to stimulate the release of pre-ecdysis and ecdysis-triggering hormones from the Inka cells of M. sexta, inducing precocious pre-ecdysis and ecdysis behaviors [10]. In the present study, we wanted to fully characterize corazonin in a hymenopteran insect. The Hymenoptera are one of the largest and economically most important insect orders. Apis mellifera in particular is a social insect and a model organism to study development, immunity and behavior.
2.
Material and methods
2.1.
Insects
2.2.
The Quickprep Micro mRNA Purification Kit (Amersham Biosciences, USA) was used to isolate the mRNA of 50 CNSs of Ap. mellifera workers. cDNA was made with the SuperScript First-Strand Synthesis System (Invitrogen, USA). The ORF of the Apis corazonin precursor was amplified by the Advantage 2 PCR Enzyme System (BD Biosciences, USA) with specific oligonucleotide PCR primers (Eurogentech, Belgium). Based on the Apis ESTdatabase [32] the forward primer was 50 -CCA GAT GGT TAA CTC TCA AAT CC-30 and the reverse primer 50 TTA ATA ATT GTT ATT GGT AGG AGC G-30 (Fig. 1, bold type). The PCR was conducted with the following parameters: first a denaturation step of 94 8C for 60 s, then 30 cycles of 95 8C for 30 s and 58 8C for 30 s, and finally an extra extension step of 68 8C for 60 s. The obtained PCR product was first cloned in the pCR4-TOPO vector using the TOPO-TA cloning kit (Invitrogen, The Netherlands). Then the vector was transformed into E. coli TOP10 (Invitrogen, The Netherlands) which were grown overnight at 37 8C in a shaking incubator. After selection of the obtained clones, the plasmid DNA was isolated with the Nucleospin Plasmid Quick Pure kit (Macherey-Nagel, Germany) and digested with EcoRI. Prior to DNA sequence analysis (310 Genetic Analyzer, Applied Biosystems, UK), the precursor DNA was amplified with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit v1.1 (Applied biosystems, UK). The PCR parameters were: 96 8C for 30 s, 50 8C for 15 s and 60 8C for 240 s (25 cycles).
2.3. Foraging workers of the honey bee Ap. mellifera carnica (Hymenoptera) were obtained from a local beekeeper. Workers of the wasp Vespula saxonica were caught daily upon emergence from an isolated nest. Workers of the red wood ant Formica rufa were caught from a large nest in Heverlee (Belgium). Albino Locusta migratoria from the Okinawa strain were reared in crowded conditions at an average temperature of 29 8C, a photoperiod of 13-h light:11-h dark and a diet of grass and oat flakes.
Cloning of the corazonin precursor in Ap. mellifera
Mass spectrometry
Forty retrocerebral complexes (corpora cardiaca and corpora allata) of Ap. mellifera were dissected for tandem MS analysis. Thirty retrocerebral complexes of Ap. mellifera, F. rufa and V. saxonica were dissected for MS analysis. Tissues were dissected and rinsed in physiological saline and extracted in 100 mL methanol/water/formic acid (FA) (90:9:1, v/v/v). The extracts were sonicated and centrifuged at 10,000 rpm for 10 min. The pellets were extracted, sonicated and centrifuged
Fig. 1 – Nucleotide sequence and the deduced amino acid sequence for the open reading frame of the cDNA encoding the Apis mellifera corazonin precursor. The sequences corresponding to the oligonucleotide primers used in the PCR amplification are in bold. The amino acid sequence reveals a signal peptide (italics), followed by corazonin (underlined), a dibasic cleavage site and the corazonin related peptide.
peptides 27 (2006) 493–499
a second time. The pooled supernatants of 40 Ap. mellifera retrocerebral complexes were filtered through a Millipore spindown filter, dried and stored at 20 8C. Capillary LC– tandem MS was performed as described [28]. In brief, 10 mL of the sample (containing an equivalent of 15 retrocerebral complexes) was loaded onto a pre-column with an isocratic flow of 2% CH3CN in water with 0.1% FA at a flow rate of 10 mL/ min. After 2 min, the pre-column was switched online with the analytical capillary column at a flow rate of 150 nL/min. Ions with m/z close to 662.23 (which is the calculated mass of Ap. mellifera corazonin (1322.57 Da) +2H+/2z) were automatically selected for fragmentation during the nano–LC tandem MS separation. The pooled supernatants of the retrocerebral complexes of Ap. mellifera, F. rufa and V. saxonica were completely evaporated and then redissolved in 25 mL water/CH3CN/trifluoroacetic acid (TFA) (97.9:2:0.1, v/v/v). A ZipTipC18 (Millipore, 15 mm) was used to concentrate and desalt the sample. Samples were eluted with 3 mL water/CH3CN/FA (69.9:30:0.1, v/v/v). One or two microlitres of the samples were transferred to a ground steel target plate and mixed with 0.5 mL a-cyano-4-hydroxycinnamic acid in acetone, air-dried and analyzed by MALDITOF MS as described [31].
2.4.
495
coli, DNA sequence analysis revealed the Apis corazonin precursor (Fig. 1). The corazonin precursor contains a signal peptide of 21 amino acid residues, followed by corazonin, a dibasic cleavage site and a corazonin associated peptide. Apis corazonin differs from [His7]-corazonin by the substitution of glutamine4 by a threonine residue (Fig. 1). Hence, Apis corazonin is the third isoform of this neuropeptide. An ion peak with a mass that matches Apis corazonin can be observed in a MALDI spectrum of retrocerebral complexes (Fig. 2A). However, the presence of this isoform in these endocrine organs of Ap. mellifera was unequivocally confirmed by means of tandem MS (Fig. 3). Note that when total brains were dissected, the ion peak corresponding with Apis corazonin
Dark color inducing bioassay
Albino Locusta migratoria from the Okinawa strain were collected after the molt to the fourth instar. Groups of 6 (control) to 12 individuals were injected on day 0 with 3 mL of rapeseed oil containing 1 nmol, 10 or 0.1 pmol of synthetic Apis corazonin. A negative control group was injected with pure rapeseed oil. As a positive control, locusts were injected with similar amounts of [His7]-corazonin. The color of the locusts was evaluated 2 days after injection and 2 days after they molted to the fifth instar. No darkening at all was observed in the pure rapeseed oil injected locusts, whereas the locusts injected with [His7]-corazonin showed a similar concentration dependent grade of black coloration as described [33].
2.5.
Whole mount immunohistochemistry
Polyclonal antisera against [His7]-corazonin were raised in New Zealand White rabbits, which were immunized with synthetic [His7]-corazonin that was conjugated to thyroglobulin by means of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and glutaraldehyde. The specificity of the antiserum was demonstrated using pre-immune antiserum. The primary antiserum was pre-absorbed with 100 mM of the synthetic peptide. The CNS of Ap. mellifera foragers was studied using the whole mount protocol as described [28].
3.
Results
3.1.
A third bioactive isoform of corazonin in Ap. mellifera
Amplification of the cDNA, derived from 50 CNSs of Apis mellifera, with the specific oligonucleotide primers resulted in a PCR product of the calculated length (slightly above 300 bp). After cloning in the pCR4-TOPO vector and transfection in E.
Fig. 2 – MALDI-TOF mass spectra from an equivalent of 10 to 20 retrocerebral complexes of the honey bee Ap. mellifera (A), the red wood ant F. rufa (B) and the wasp V. saxonica (C). Monoisotopic [M + H+] are given. An ion peak mass corresponding with the calculated mass of [Thr4, His7]-corazonin (M = 1322.57) was found in Ap. mellifera (A). An ion peak mass matching the calculated mass of [Arg7]-corazonin (M = 1368.62) was found in F. rufa (B). A small ion peak mass corresponding with the calculated mass of [His7]-corazonin (M = 1349.58) was found in V. saxonica (C).
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Fig. 3 – CID (collision induced dissociation) spectrum of pQTFTYSHGWTNamide or Apis Corazonin. a-type, b-type, y-type fragment ions are indicated. NanoLC run of 15 retrocerebral complexes of Apis mellifera.
could not be discerned (data not shown). This is most probably due to a lower concentration of corazonin in the brain. Injection of 1 nmol of synthetic Apis corazonin in albino locusts resulted in a complete darkening 2 days after the moult to the fifth instar, whereas 10 pmol caused darkening of the lowest degree in some individuals and 0.1 pmol had no effect at all (data not shown). These results are in agreement with the findings of Yerushalmi et al. who stated that the full sequence of [His7]-corazonin is required for a maximal effect [33]. Ten picomoles of the entire [His7]-corazonin sequence resulted in a complete darkening of most locusts, but an equal amount of N- or C-terminal shortened sequences had only minor effects [33].
3.2.
Corazonin immunoreactivity in Ap. mellifera
Whole mount immunohistochemistry was used to investigate the pattern of corazonin immunoreactivity in the CNS of Ap. mellifera. Pre-immune antiserum and pre-absorbed primary
antiserum did not cause any staining (not shown). Using this primary corazonin antiserum, four pairs of neurosecretory cells were observed in the lateral protocerebrum (Fig. 4). These neurons project parallel processes into the medial protocerebrum and send one bundle of axons posteriorly and ventrolaterally. This bundle turns towards the midline before leaving the brain and innervating the ipsilateral retrocerebral organs of Ap. mellifera (Fig. 5), in which corazonin was detected by means of mass spectrometry (Figs. 2A and 3). The presence of corazonin-like immunoreactivity in two groups of protocerebral lateral neurosecretory cells projecting into the ipsilateral retrocerebral neurohemal organs is consistent with a distribution pattern observed in all insect species investigated so far, except for beetles [2,15,17–19,27]. The presence of corazonin-like immunoreactive cells in the thoracic and abdominal ganglia on the other hand, shows much more variation [2,17,27]. In situ hybridization in Ga. mellonella confirmed the presence of corazonin in the lateral neurosecretory cells, whereas no corazonin precursor mRNA could be detected in other cells of the nervous system [5]. We found no obvious corazonin-like immunoreactive cells in the thoracic nor in the abdominal ganglia of Ap. mellifera.
3.3.
Fig. 4 – Detail of the corazonin immunoreactivity in the pars lateralis of a whole mount tissue preparation of the adult brain of Apis mellifera. This image was obtained by superimposing seven optical sections 1.5 mm apart. Immunoreactivity was found in four neurons with axons projecting posteriorly. Bar = 50 mm.
Is Apis corazonin the sole isoform in Hymenoptera?
A previous study has shown by means of the dark color inducing bioassay, that besides Ap. mellifera other Hymenoptera also contain a bioactive corazonin-like substance [20]. The question arises now whether or not Apis corazonin counts in particular for the dark color inducing activity of the other hymenopteran species investigated [20]. This question can be represented as: is Apis corazonin the sole isoform throughout all the species of this insect order? Therefore, we used MALDITOF mass spectrometry to scan the retrocerebral complexes of two other relatively large hymenopteran species, i.e. the wasp V. saxonica and the red wood ant F. rufa for the presence of the typical bee [Thr4, His7]-corazonin. The ion mass corresponding with this corazonin (M = 1322.57 Da) could, in contrast to Ap. mellifera (Fig. 2A), not be detected in the MALDI-TOF mass spectra of the retrocerebral complex in case of F. rufa and V. saxonica (Fig. 2B and C). However, the mass spectrum of the brain-CC extract of F. rufa shows a large peak with an ion mass corresponding to the calculated mass of [Arg7]-corazonin (M = 1368.62 Da), indicating that this isoform is present in this
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to have originated late during evolution in different insect orders. The presence of corazonin-like substance in the primitive insect, the silverfish Ctenolepisma lineata [17] and even in the crustacean species, the Jonah crab Cancer borealis [11] suggests an ancient origin of this neuropeptide. In this perspective, other corazonin isoforms might be out there and beetles might not have lost corazonin but might contain a dramatically changed isoform that is inactive in the dark color inducing bioassay.
Fig. 5 – Corazonin immunoreactivity in the adult brain of Apis mellifera. Schematic drawing based on whole mount tissue preparations. Frontal view, immunoreactivity is bilaterally symmetrical, the immunoreactivity of the left hemisphere is depicted. Immunoreactivity was found in four neurons with processes into the medial protocerebrum. One bundle of axons innervates the ipsilateral corporus cardiacum (CC) via the posterior lateral tract (PLT). Esophageal opening (EF); subesophageal ganglion (SG).
ant species (Fig. 2B). The mass spectrum of the brain-CC extract of V. saxonica on the other hand, contains a small but distinct ion peak with the calculated mass of [His7]-corazonin (M = 1349.58) providing evidence that [His7]-corazonin or perhaps a fourth isoform might be present in this wasp species (Fig. 2C). Hence, our findings indicate that Apis corazonin is not present in all Hymenoptera. The presence of a particular corazonin isoform is thus not determined by the insect order. In other words, several corazonin isoforms seem
4.
Discussion
4.1. The conservation of corazonin and its precursor throughout insect species and orders Only three insect neuropeptide precursors, i.e. the adipokinetic hormone (AKH), SIFamide and corazonin precursors display a similar general structural pattern: one copy of the bioactive peptide is situated immediately after the signal peptide and separated by the common GKR processing site from the subsequent joining peptide. In addition, the Akh, Crz and IFa genes of at least D. melanogaster have in common that they are characterized by two exons separated by a small intron (www.flybase.org). Small sequence similarities between AKH and corazonin suggested an evolutionary link [24]. This hypothesis was confirmed by the fact that the Drosophila AKH and corazonin receptors are structurally related, which even implies a co-evolution of ligands and receptors [3,13]. Furthermore, the joining peptides of AKH have cysteine residues allowing the formation of dimers via cystine bridges, as was demonstrated in the locusts S. gregaria and L. migratoria [6,9]. Recently, it was found that all known
Fig. 6 – Alignment of insect corazonin precursors. Gam is Galleria mellonella, accession AY004866.1 (AAF87082.1); Bom Bombyx mori, accession AB106876.1 (BAC66443.1); Drm Drosophila melanogaster, accession NM_079626.2 (NP_524350.1; CG3302); Drp Drosophila pseudoobscura, accession AADE01000000 (AADE01000051); Apm Apis mellifera (gnljAmel_1.1jGroupUn.323); Aea Aedes aegypti (TC35312) and Ang is Anopheles gambiae, accession XM_313456.1 (ENSANGP00000020275). Sequences were retrieved by BLAST searching and processing the available genomic databases of insect species. CLUSTALW multiple alignment (www.ebi.ac.uk/clustalw) was performed. Signal peptides predicted by the SignalP program (http://www.cbs.dtu.dk/services/SignalP/[12]) are in italics. Identical amino acid residues in bold. Dibasic cleavage sites and cysteine residues are underlined.
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joining peptides of the SIFamide precursor in insect species have two cysteine residues, separated by six amino acid residues [30]. Dimerization of the relatively well conserved SIFamide precursors is thus possible as well. To investigate whether corazonin precursors contain cysteine residues, the currently available genomic databases of insects were extensively datamined and processed. The corazonin precursors of seven insect species were detected and compared (Fig. 6). The corazonin precursor of D. melanogaster was already cloned in 1994 from a genomic library [26]. A corazonin-precursor-related peptide (CPRP) of 39 amino acid residues, with an internal potential dibasic cleavage site (RR) but without cysteine residues was predicted [26]. However, an intron between amino acid residues 72 and 73 and containing a TAA stopcodon was overlooked [1]. Without this intron the Drosophila corazonin precursor does contain two cysteine residues (Fig. 6). A similar intron at about the same position was also found in the corazonin genes of Ap. mellifera, B. mori and D. pseudoobscura (data not shown). The corazonin precursors of G. mellonella and Ae. aegypti were derived from cDNA, thus it is unknown whether these corazonin genes have a similar organization. Interestingly, no such intron could be predicted in the corazonin gene of the malaria mosquito An. gambiae. Moreover, the originally annotated Anopheles corazonin precursor (Fig. 6) was replaced by a precursor containing only the four N-terminal amino acids of this precursor, which seems highly improbable. Nevertheless, all corazonin precursors display at least one cysteine residue, although the corazonin joining peptide is clearly not as conserved as it is the case for SIFamide joining peptide and AKH joining peptide. Thus, dimerization or internal cystine bridges cannot be excluded. In addition, all corazonin precursors, except for the Galleria precursor, contain at least one internal dibasic cleavage site, allowing further processing of the corazonin joining peptide (Fig. 6).
5.
Conclusion
In conclusion, we characterized a third isoform of corazonin in the honey bee Ap. mellifera, which is most likely not present in all Hymenoptera. Like [His7]- and [Arg7]-corazonin, Apis corazonin is biologically active in the locust coloration assay, although to a lesser extent. Apis corazonin is present in a group of lateral cells in the protocerebrum which innervate the retrocerebral complex. The Apis corazonin joining peptide shows minor sequence similarities with the other identified corazonin joining peptides, although the presence of cysteine residues seems conserved, as it is the case in the joining peptides of the related neuropeptides AKH and SIFamide.
Acknowledgements P. Verleyen and I. Mertens are post-docs of the KULeuven Research Council, G. Baggerman and J. Huybrechts are postdoctoral researchers of the Fund for Scientific ResearchFlanders (Belgium) (F.W.O.-Vlaanderen). Research was supported by the FWO-grants G.0175.02 and G.O146.03. We thank Mrs. Chris Heremans for the supply of Apis mellifera, Dr. Eric Schoeters and the ALRC for their assistance.
references
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