Genomics and Peptidomics of Neuropeptides and Protein Hormones

Present in the Parasitic Wasp Nasonia vitripennis|

Frank Hauser,*,† Susanne Neupert,‡ Michael Williamson,† Reinhard Predel,‡ Yoshiaki Tanaka,§
and Cornelis J. P. Grimmelikhuijzen†

Center for Functional and Comparative Insect Genomics, Department of Biology, University of Copenhagen,
DK-2100 Copenhagen, Denmark, Institute of Zoology, University of Jena, D-07743 Jena, Germany, and
National Institute of Agrobiological Science, Division of Insect Science, Tsukuba, Ibaraki 305-86 34, Japan

Received June 8, 2010

Neuropeptides and protein hormones constitute a very important group of signaling molecules,
regulating central physiological processes such as reproduction, development, and behavior. Using a
bioinformatics approach, we screened the recently sequenced genome of the parasitic wasp, Nasonia
vitripennis, for the presence of these signaling molecules and annotated 30 precursor genes encoding
51 different mature neuropeptides or protein hormones. Twenty-four of the predicted mature Nasonia
neuropeptides could be experimentally confirmed by mass spectrometry. We also discovered a
completely novel neuropeptide gene in Nasonia, coding for peptides containing the C-terminal sequence
RYamide. This gene has orthologs in nearly all arthropods with a sequenced genome, and its expression
in mosquitoes was confirmed by mass spectrometry. No precursor could be identified for N-terminally
extended FMRFamides, even though their putative G protein coupled receptor (GPCR) is present in the
Nasonia genome. Neither the precursor nor the putative receptor could be identified for allatostatin-B,
capa, the glycoprotein hormones GPA2/GPB5, kinin, proctolin, sex peptide, and sulfakinin, arguing that
these signaling systems are truly absent in the wasp. Also, antidiuretic factors, allatotropin, and NPLP-
like precursors are missing in Nasonia, but here the receptors have not been identified in any insect,
so far. Nasonia (Hymenoptera) has the lowest number of neuropeptide precursor genes compared to
Drosophila melanogaster, Aedes aegypti (both Diptera), Bombyx mori (Lepidoptera), Tribolium
castaneum (Coleoptera), Apis mellifera (Hymenoptera), and Acyrthosiphon pisum (Hemiptera). This
lower number of neuropeptide genes might be related to Nasonia’s parasitic life.

Keywords: neuropeptide • protein hormone • PTTH • GPCR • genomics • peptidomics • MALDI-TOF •

CID • mass spectrometry • insect

Introduction In addition to the three Nasonia species, the genomes of at

The tiny parasitic wasp Nasonia vitripennis (Hymenoptera:
Pteromalidae) has been used for genetic, ecological, evolution-
ary and developmental research for many decades.1,2 Nasonia
lays eggs into the pupae of various fly species, primarily
blowflies and fleshflies, and this interesting life style has
stimulated research on host finding, host selectivity, and many
other aspects of parasitic behavior.3 The usefulness of parasitic
wasps for the biological control of pest insects together with
its potentials as a model insect for biological research have
recently led to the sequencing of the genome of N. vitripennis
and two other parasitic wasps belonging to the genus Nasonia.4
|
The cloned cDNA sequences reported in this paper have GenBank
accession numbers GU937435, HM461994 and HM461995.
* To whom correspondence should be addressed. F. Hauser, Center for
Functional and Comparative Insect Genomics, Department of Biology,
Universitetsparken 15, DK-2100 Copenhagen, Denmark; Tel. +45 3532 1206;
Fax +45 3532 1200; e-mail: fhauser@bio.ku.dk.
†
University of Copenhagen.
‡
University of Jena.
§
National Institute of Agrobiological Science.
least 25 other insect species have been sequenced or are in
the pipeline to be sequenced5 (http://www.ncbi.nlm.nih.gov/
sutils/genom_table.cgi?organism)insects). These insects be-
long mainly to the Holometabola (insects with a complete
metamorphosis), of which they represent all major orders
(Diptera, Lepidoptera, Coleoptera, Hymenoptera). With the
recently sequenced genome from Nasonia we now have an
excellent tool to do comparative genomics studies, especially
because another hymenopteran, the honey bee Apis mellifera,
which in contrast to Nasonia has a social life style, also has
been sequenced recently.6
One important group of proteins to be analyzed using the
comparative genomics approach are neuropeptides and protein
hormones, which are signaling molecules that control central
physiological processes such as development, reproduction,
behavior and homeostasis. The receptors for these neuropep-
tides are usually G protein-coupled receptors (GPCRs), which
are transmembrane proteins located in the cell membrane of
the target cells.7,8

5296 Journal of Proteome Research 2010, 9, 5296–5310 10.1021/pr100570j  2010 American Chemical Society
Published on Web 08/09/2010
Global Analysis of Neuropeptides in Nasonia
The availability of the sequenced Nasonia genome now
allows us to identify the complete set of neuropeptides and
protein hormones in this organism and to compare this set
with those recently found in the genomes from the honeybee
A. mellifera,9,10 the fruit fly Drosophila melanogaster,11,12 the
mosquito Aedes aegypti,13 the silkworm Bombyx mori,14 the red
flour beetle Tribolium castaneum,15,16 and the pea aphid
Acyrthosiphon pisum.17 This comparative genomics approach
should give us valuable information to understand the evolu-
tion of neuropeptides, and if possible to correlate insect life
style with neuropeptide gene content.
Here, we searched the Nasonia genome with known insect
neuropeptide and protein hormone precursors and identified
30 precursor genes encoding 51 different putative mature
neuropeptides or protein hormones. The presence and struc-
ture of 24 of these neuropeptides could be confirmed by direct
peptide profiling of brain tissue by MALDI-TOF mass spec-
trometry (MS). Because neuropeptides and protein hormones
require specific receptors to function in the organism, we have,
in parallel, also identified all Nasonia neuropeptide and protein
hormone receptors belonging to the GPCR superfamily (Hauser
et al., unpublished). The presence or absence of a neuropeptide
receptor in the analyzed Nasonia genome further supports our
current findings of the presence or absence of the neuropeptide
and protein hormone genes.
Experimental Section
Genomics and Software. The whole genome sequence of
Nasonia vitripennis, version 1.0, was used for homology
searches with known invertebrate neuropeptide sequences at
the Web site of the Human Genome Sequencing Centre, Baylors
College of Medicine (http://blast.hgsc.bcm.tmc.edu/blast.hgsc),
using TBLASTN. The assembled version 1.0 covers 98% of BAC
and 97% of EST sequences. In the remaining unassembled trace
sequences, we could not detect any neuropeptide genes. For
TBLASTN homology searches in other species, the nucleotide
collection, nonhuman nonmouse ESTs or whole-genome shot-
gun reads databases at NCBI (http://blast.ncbi.nlm.nih.gov/
Blast.cgi) were chosen. Prediction of the gene structure and
open reading frame was done in GENBOREE (http://www.
genboree.org/java-bin/login.jsp) that contains the GLEAN2
predictions incorporating the results from multiple gene pre-
diction programs and by manual correction. Signal peptides
were predicted by the SIGNALP server (http://www.cbs.dtu.dk/
services/SignalP/). Multiple sequence alignments were per-
formed in ClustalW (http://www.ebi.ac.uk/Tools/clustalw/), or
using the Lasergene software package (DNASTAR inc.) with
manual adjustments. Phylogenetic analyses were done, using
Lasergene software. Peptide processing was predicted using the
ProP program (http://www.cbs.dtu.dk/services/ProP/).
Dissection and Sample Preparation for Mass Spectrometry.
N. vitripennis (adults and larvae) were fixed with microneedles
and submerged in insect saline (pH 7.25) of the following
composition: NaCl (7.50 g/L), KCl (0.20 g/L), CaCl2 (0.20 g/L)
and NaHCO3 (0.10 g/L). The body cavity or the head capsule
was opened with fine forceps and ultrafine scissors. Different
ganglia of the ventral nerve cord, brain/subesophageal ganglion
(see Supporting Information, Figure S1), and corpora cardiaca
were dissected (at least five preparations of each tissue sample)
and cut in smaller pieces, which were transferred with a glass
capillary into a drop of distilled water on the sample plate for
MALDI-TOF MS. The water was subsequently removed using
a glass capillary. Subsequently, the tissue was air-dried and
research articles
covered with approximately 50 nL of matrix solution (saturated
R-cyano-4-hydroxycinnamic acid dissolved in methanol/water
1:1, v/v) over a period of about 5 s using a Nanoliter injector
(World Precision Instruments, Berlin, Germany). Each prepara-
tion was air-dried again and finally covered with destilled water
for a few seconds, which was removed by cellulose paper.
MALDI-TOF MS. MALDI-TOF analyses were performed on
an ABI 4800 Proteomics Analyzer (Applied Biosystems, Framing-
ham, MA). To determine the parent masses, the instrument
was initially operated in reflectron mode. For the tandem MS
experiments (performed in gas off mode), a CID acceleration
of 1 kV was used in all cases. The number of laser shots used
to obtain a spectrum varied from 800-4000, depending on
signal quality. The fragmentation patterns were used to manu-
ally determine the sequence of the peptides by using the Data
ExplorerT software package.
cDNA Cloning. For PCR, total RNA was isolated from whole
adult animals (mixed males and females), using the NucleoSpin
RNA II kit (Macherey-Nagel). cDNA was synthesized and
amplified using the FirstChoice RLM-RACE kit (Ambion).
Primers are given in the Supporting Information section Table
S1.
Results
Table 1 gives an alphabetical list of the 51 different neu-
ropeptides that are encoded by the 30 neurohormone precursor
genes we identified in the Nasonia genome (for FASTA-
sequences of the precursors see Supporting Information, Figure
S2). Only 12 of these precursor genes have been automatically
identified as genes by the genome project software, whereas
the other 18 have been manually identified by us. Table 2
divides these 30 precursor genes into two different groups, A
or B. For all members of group A, a likely receptor gene could
be identified in Nasonia, giving additional support for the
presence of these neuropeptides in Nasonia. For members of
group B: a receptor gene can not be given, because these
receptors have not been identified (deorphanized) yet in any
insect.7,8 The neuropeptides that we could not identify, al-
though we found a putative receptor in Nasonia, are given in
group C. In group D, absent neuropeptides are shown for which
we also could not find their receptors. These results, therefore,
confirm that these peptides are truly absent in Nasonia. Finally,
group E lists absent peptides for which a receptor is unknown
in any insect. Below, we discuss these groups in more details.
A. Neuropeptides or Protein Hormones That Can Be
Matched with Putative Receptors. We found 22 wasp neu-
ropeptide and protein hormone precursor genes that could be
matched with putative receptors (Table 2, Group A). Products
of seven of these neuropeptide genes (adipokinetic hormone,
allatostatin-A, allatostatin-C, corazonin, myosuppressin, orco-
kinin, and short neuropeptide F) were detected in the corpora
cardiaca, using mass spectrometry (both MALDI-TOF and CID),
which suggests that they are neurohormones (Figure 1). Ad-
ditional neuropeptides were identified by mass spectrometry
in specific brain regions such as pars intercerebralis, accessory
medulla, and antennal lobe (Figure 2, Figure 3), or in the
thoracic ganglia (Figure 4).
ACP. ACP (adipokinetic hormone/corazonin-related peptide)
and its receptor represent a recently discovered novel insect
signaling system closely related to, but functionally indepen-
dent from the insect adipokinetic hormone (AKH) and cora-
zonin systems.18 The Nasonia ACP precursor encodes a single
putative neuropeptide, the decapeptide pQVTFSKGWGPamide
Journal of Proteome Research • Vol. 9, No. 10, 2010 5297
research articles Hauser et al.
Table 1. Alphabetical List of Mature Neuropeptides and Protein Hormones Present in Nasonia

a b
For longer peptide sequences, the accession no. assigned by the Nasonia Genome Project is given. Allatostatin-A-4 and RYamide-1 are only
confirmed by mass match; ion intensity was not sufficient for fragment analysis.

5298 Journal of Proteome Research • Vol. 9, No. 10, 2010

Global Analysis of Neuropeptides in Nasonia research articles
Table 2. Neurohormone Precursors Present (A and B) or Absent (C-E) in Nasonia

a b
nd, not detected in Nasonia; na, not applicable (no receptor known in any insect). The cDNAs of these genes have been cloned (Figures S3-S5,
Supporting Information).

(Table 1 and Supporting Information, Figure S2). In Figure 5,
the sequence of the Nasonia ACP precursor is aligned with ACP
precursors found in other insects. This alignment clearly shows
the similar overall structure of all insect ACP precursors, where
the ACP sequence is directly located after the signal peptide
sequence. Also the C-terminal parts of the precursors contain
several highly conserved residues, including a potential cystine
bridge. This organization resembles that of the AKH and corazonin
precursors.18 Interestingly, ACP and its receptor are absent in
Drosophila, honey bee, pea aphid and the body louse.18

Journal of Proteome Research • Vol. 9, No. 10, 2010 5299

research articles
Figure 1. MALDI-TOF mass spectrum typical of a preparation of
a single pair of corpora cardiaca from Nasonia. The assigned
neuropeptides are labeled and the identity was subsequently
confirmed by fragment analysis (not shown). The presence of
these neuropeptides in the corpora cardiaca suggests a role as
neurohormones. Not all ion signals can be assigned to products
of annotated neuropeptide genes. Detection of only a single
orcokinin is in accordance with the copy number of ORC-2 (6×)
in the orcokinin preprohormone. The other orcokinins were
detectable in spectra with higher orcokinin abundance (see Figure
2). We do not regard NVP as a neuropeptide, because, although
originally identified in the honeybee,9 this peptide sequence is
not conserved in insects (see Supporting Information, Figure S6).
The NVP signal shown in this and other mass spectra comes from
a peptide without the NVP sequence located in a region of the
NVP protein different from the NVP region in the honeybee
protein (see regions highlighted in red and green in Supporting
Information, Figure S6). AKH, adipokinetic hormone; AST, alla-
tostatin; AST-PP, allatostatin precursor peptide (AYTYRSEY-OH);
CC, corpora cardiaca; COR, corazonin; MS, myosuppressin; ORC,
orcokinin; sNPF, short neuropeptide F.
AKH. Adipokinetic hormones (AKHs) are 8 to 10 amino acid
residues long insect neuropeptides expressed in endocrine cells
from the corpora cardiaca.19 They control lipid and sugar
mobilization from the fat body during energy-consuming
activities such as flight and intense locomotion. In the Nasonia
genome, a single putative AKH precursor gene was found
encoding the peptide pQLNFSTGWamide (Table 1 and Sup-
porting Information, Figure S2). We confirmed the existence
of this hormone in Nasonia by cDNA cloning of the entire AKH
prepropeptide (Supporting Information, Figure S3) and by MS
(Figure 1, Table 1).
Allatostatin-A. In various insects, type A allatostatins inhibit
the corpora allata to produce juvenile hormone or block muscle
contraction in the gut.11 The Nasonia allatostatin-A precursor
encodes 5 putative amidated peptides with the common
C-terminal motif Y/FXFGLamide (Table 1 and Supporting
Information, Figure S2). Four of these peptides and an ad-
ditional precursor peptide (AYTYRSEY-OH) could be con-
firmed by MS (Figure 1, Table 1). Due to the lack of amino
acids with charged side chains, AST-A-1, -2, and -4 show a very
low signal intensity in mass spectra and only the [M + Na/K]+
adduct ions of these peptides were detectable. The nondetec-
tion of the predicted AST-A-5 suggests that the predicted
monobasic cleavage site (Lys) preceding the N-terminus is not
being used.
Allatostatin-C and Allatostatin-CC. Another insect neu-
ropeptide inhibiting the juvenile hormone production of the
5300 Journal of Proteome Research • Vol. 9, No. 10, 2010
Hauser et al.
Figure 2. MALDI-TOF mass spectra typical of preparations from
specific brain regions. (A) Pars intercerebralis of the protocer-
ebrum. In addition to peptides observed in the corpora cardiaca,
ion signals of tachykinins (TK), SIFamide, and diuretic hormone
(DH-31) can be identified. (Inset) Different signal intensities of
nearly identical orcokinins which originate from different copy
numbers in the preprohormone. (B) Accessory medulla of the
optic lobes with a number of neuropeptides, including weak
signals of pigment dispersing factor (PDF). We do not regard ITG
as a neuropeptide, because, although originally identified in the
honeybee,9 this peptide sequence is not conserved in most other
insects, although the protein itself is conserved (Figure S7,
Supporting Information). The ITG peptide sequence shown in this
and other mass spectra is highlighted in green in Figure S7
(Supporting Information).
corpora allata (for example in the moth Manduca sexta) is
allatostatin-C, a cyclic neuropeptide that is structurally unre-
lated to allatostatin A.11,20-22 In Nasonia, there is an allatostatin
C gene coding for the peptide NYWRQCAFNAVSCFamide
(Table 1 and Supporting Information, Figure S2). By mass
spectrometry, this peptide was found throughout the CNS and
also in the corpora cardiaca (Figures 1-3). Nasonia allatosta-
tin-C is very similar to allatostatin-C found in the honey bee
A. mellifera (Hymenoptera, Holometabola) and several hemi-
metabolous insects (all amidated at the C-terminus), but quite
different from the nonamidated allatostatins-C occurring in
holometabolous insects other than the hymenopterans.22
In addition to this allatostatin-C precursor, a partial precur-
sor gene coding for an allatostatin-C paralog, allatostatin-CC,
has recently been annotated in Nasonia and other insects22
(Table 1). The function of allatostatin-CC is still unknown.22
To confirm the existence of allatostatin-C and allatostatin-CC
in Nasonia, we cloned the cDNAs encoding the entire prepro-
peptides of both paralogs (Supporting Information, Figure S4
and S5).
Bursicon. The molting glycoprotein hormone bursicon is,
like all mammalian glycoprotein hormones, a heterodimer
composed of two larger N-glycosylated cystine-knot polypep-
tides, alpha- and beta-bursicon.23,24 Bursicon causes tanning
and hardening of the soft cuticle after adult ecdysis and induces
wing expansion and apoptosis of the wing epithelial cells after
Global Analysis of Neuropeptides in Nasonia
Figure 3. Mass spectrum analysis of neuropeptides in the
antennal lobe. (A) MALDI-TOF mass spectrum typical of a
preparation of a single antennal lobe of Nasonia (approximately
50 µm in length). The assigned neuropeptides are likely involved
in the processing of the olfactory information: sNPF, short
neuropeptide F; AST, allatostatin; TK, tachykinin. Particularly
impressive in this context is the abundance of TK 1-5. (B) CID
mass spectrum of TK-2 at [M + H]+: 993.4, taken from the same
sample. Fragment ions are labeled and confirm the amino acid
sequence of TK-2.
Figure 4. MALDI-TOF mass spectrum of a preparation of a
thoracic ganglion from Nasonia. The arrow marks a substance
that is mass-identical with a putative product of the RYamide
preprohormone (pQDNFYASGRFamide). Due to low signal in-
tensity, it was not possible to perform fragment analysis of this
peptide.
research articles
25-27
completed expansion. In the Nasonia genome, we found
one precursor for alpha- and one for beta-bursicon. Both
glycoprotein subunits contain the typical pattern of 11 cystein
residues that can form 5 intramolecular and one intermolecular
disulfide bridges (Supporting Information, Figure S2).
CCAP. The crustacean cardioactive peptide CCAP
(PFCNAFTGCamide) is a highly conserved cyclic neuropeptide
originally isolated from the shore crab Carcinus maenas, but
present in identical form also in insects.11 It is coexpressed with
bursicon in several neurons of the insect nervous system,
especially in the abdominal ganglia, where it might support
the action of bursicon.25,28 CCAP is a multifunctional peptide
that has cardioexcitatory and AKH-releasing activity, but also
controls the motor behavior program associated with ecdysis.29
The Nasonia CCAP precursor gene encodes, like in other
insects, a single copy of this neuropeptide (Table 1 and
Supporting Information, Figure S2).
Corazonin. Corazonin was originally isolated from cock-
roaches based on its cardio-excitatory actions on the isolated
cockroach heart.30 In locusts, corazonin induces a darkening
of the exoskeleton in association with swarm formation and
migration.31 Furthermore, corazonin is proposed to be associ-
ated with behavioral and physiological responses to stress.32-34
Corazonin is found in most insects, but not in Coleoptera and
the pea aphid.16,17,35,36 Within the Hymenoptera, at least four
different isoforms of corazonin occur which is the greatest
diversity found for this peptide in any insect order.35 In
Nasonia, we found one precursor gene that encodes the peptide
[Arg7]-corazonin (pQTFQYSRGWTNamide, Supporting Infor-
mation, Figure S2), which is identical to the ancient corazonin
found in most insects and also in crustaceans.35 The expression
and structure of this peptide could be confirmed by MS (Figure
1, Table 1). Thus, the remarkable sequence variation of cora-
zonin in the Hymenoptera seems to be restricted to the
aculeatan lineage (“stinging wasps”) of Hymenoptera.
DH31 and DH44. In Drosophila, two structurally unrelated
diuretic hormones, DH31 (31 amino acid residues long) and
DH44 (44 amino acid residues long), act on the Malpighian
tubules to control water homeostasis.37 DH31 is structurally
related to mammalian calcitonin, whereas DH44 resembles
corticotropin-releasing factor (CRF).38 In Nasonia, we an-
notated one orthologue to each of these hormones (Table 1
and Supporting Information, Figure S2). The Nasonia DH31
could be confirmed by MS (Figure 2, Table 1).
EH. Eclosion hormone (EH) is produced by a single pair of
brain neurons during each insect molt.39 In the central nervous
system, EH induces the release of CCAP, whereas it acts
peripherally on Inka cells in the epitracheal glands and induces
the release of ecdysis triggering hormone.40 Recently, a mem-
brane-bound guanylyl cyclase was identified as an EH receptor
in Inka cells.41 In Nasonia, we identified a precursor gene
coding for an EH ortholog (Table 1 and Supporting Information,
Figure S2). Interestingly, an alignment of EH sequences from
various insects shows that the Nasonia EH contains only two
of the three disulfide bridges usually present in EHs (Figure
6).42,43
ETH. In insects, ecdysis triggering hormone (ETH) is ex-
pressed in Inka cells and initiates molting.44 We found one
precursor gene coding for a single ETH orthologue with the
sequence DEPPAFFLKIAKNIPRIamide (Figure 7 and Supporting
Information, Figure S2). The overall structure of the Nasonia
ETH preprohormone resembles the ETH preprohormone found
in honey bee,9 but is different from the ETH precursors found
Journal of Proteome Research • Vol. 9, No. 10, 2010 5301
research articles Hauser et al.

Figure 5. Alignment of the ACP preprohormones from the parasitic wasp N. vitripennis (Nv-ACP, GenBank accession no. NM_001167727),
the mosquitoes A. gambiae (Ag-ACP, NM_001166025), A. aegypti (Aa-ACP, XM_001661147, manually corrected), C. pipiens (Cp-ACP,
FN391989), the silkmoth B. mori (Bm-ACP, NM_001134241), the European corn borer O. nubilalis (On-ACP, annotated from the expressed
sequence tags [EST] database accession no. GH995131), the red flour beetle T. castaneum (Tc-ACP, NM_001166025), the bloodsucking
bug R. prolixus (Rp-ACP, annotated from the whole genome shotgun sequences [WGS] database accession no. ACPB01036401) and
the black-legged tick I. scapularis (Is-ACP, annotated from WGS ABJB010491828). Amino acid residues that are common in at least
five sequences are highlighted. The immature ACP sequences are boxed. The arrows indicate the sites where signal peptidase (SP)
removes the signal peptide, or where preprohormone convertase (PC) liberates immature ACPs from the prohormone. Intron positions
are indicated by small boxes.

Figure 6. Alignment of the eclosion hormone (EH) preprohormones from N. vitripennis (Nv-EH, XM_001603287), A. mellifera (Am-EH,
XM_001122120, manually corrected), D. melanogaster (Dm-EH, NM_079662), A. aegypti (Aa-EH, XM_001652153), A. gambiae (Ag-EH,
XM_001230804), C. pipiens (Cp-EH, XM_001864393, manually corrected), B. mori (Bm-EH, NM_001043842), T. castaneum (Tc-EH,
XM_964071), A. pisum (Ap-EH, XM_001943582) and I. scapularis (Is-EH, XM_002399230). Amino acid residues that are common in at
least five sequences are highlighted. The arrow indicates the position where signal peptidase (SP) removes the signal peptide. The
conserved cysteine residues forming disulfide bonds are shown in bold. Lines below the sequences represent the position of the three
disulfide bonds usually present in EHs, lines above the sequences indicate the two putative disulfide bonds present in the Nasonia EH.

in other insects that, with the exception of pea aphid, always
contain two ETH peptides (Figure 7).45
Inotocin. Inotocin is a recently discovered insect neuropep-
tide structurally related to mammalian oxytocin and vaso-
pressin.46 Inotocin activates the identified inotocin receptor,
but otherwise the physiological role of inotocin is unknown.46
Also in Nasonia there is an inotocin peptide with the sequence
CLITNCPRLamide (Table 1). Not only the peptide sequence,
but the entire overall structure of the Nasonia inotocin pre-
propeptide resembles the mammalian preprohormones, as it
also contains a neurophysin-like sequence with 14 highly
conserved cystein residues at the C-terminal end of the
prepropeptide (Supporting Information, Figure S2).46
Myosuppressin. Myosuppressin is a decapeptide that in-
hibits contraction of a variety of visceral muscles.11 We an-
notated one precursor gene encoding a single Nasonia myo-
suppressin peptide with the sequence pQDVDHVFLRFamide
(Supporting Information, Figure S2). MS analysis confirmed the
presence and identity of this peptide in Nasonia (Figure 1,
Figure 2, Table 1). Amazingly, in the brain and corpora cardiaca
the N-terminally blocked form (pQ) and the nonblocked form
(Q) were found with about equimolar amounts (Figure 1, Figure
2).
NPF. In Drosophila, neuropeptide F is a 36 amino acid
residue long peptide with the C-terminal sequence RVRF-
amide.11 It is structurally related to the vertebrate neuropeptide

5302 Journal of Proteome Research • Vol. 9, No. 10, 2010

Global Analysis of Neuropeptides in Nasonia research articles

Figure 7. Alignment of the ecdysis triggering hormone (ETH) preprohormones from N. vitripennis (Nv-ETH, NM_001142635), A. mellifera
(Am-ETH, NM_001142607), D. melanogaster (Dm-ETH, NM_079960), A. gambiae (Ag-ETH, XM_308702), T. castaneum (Tc-ETH,
NM_001172273), B. mori (Bm-ETH, NM_001172272) and A. pisum (Ap-ETH, NM_001163212). Amino acid residues that are common in
at least three sequences are highlighted. The arrows indicate the positions where signal peptidase (SP) removes the signal peptide, or
where preprohormone convertase (PC) liberates immature peptides from the prohormone. The immature ETH sequences are boxed.
Note that Nasonia and the honey bee have only one ETH peptide, whereas higher holometabolous insects have two ETHs.

Figure 8. Alignment of the prothoracicotropic hormone (PTTH) precursors from N. vitripennis (Nv-PTTH, annotated from WGS AC185337),
B. mori (Bm-PTTH, NM_001043884), A. gambiae (Ag-PTTH, XM_555854), T. castaneum (Tc-PTTH, EEZ99381) and D. melanogaster (Dm-
PTTH, NM_134693, manually corrected). The signal peptides are highlighted in blue. Amino acid residues that are common in at least
three sequences are highlighted in gray. The conserved cysteine residues forming cystine bridges are highlighted in orange with gray
background. Lines above and below the sequences indicate the intramolecular cystine bridges, the star indicates the conserved cysteine
residues making intermolecular cystine bridges, thus forming PTTH dimers.51 Note that Nasonia has one cystine bridge less than the
other insects.

Y family and affects food intake and feeding behavior in the
fruit fly.47 In Nasonia, we annotated a gene coding for a 39
amino acid residues long NPF orthologue with the C-terminal
sequence KARYamide (Table 1 and Supporting Information,
Figure S2), which resembles NPF from the honey bee.9
PDF. Pigment dispersing factor (PDF) was first isolated from
crustaceans48 and got its name for its pigment cell dispersing
activity. Similar peptides have later been found in insects,49
where they are involved in the regulation of circadian rhythm.50
In Nasonia, there is a PDF-like precursor gene coding for the
PDF sequence NSELINSLLSLPKNMNNAamide (Supporting
Information, Figure S2). The presence and structure of this
peptide could be confirmed by MS (Figure 2, Table 1).
Prothoracicotropic Hormone (PTTH). In insects, develop-
ment and metamorphosis are coordinated by the steroid
hormones 20-hydroxyecdysone (20E). Production and release
of 20E from prothoracic glands is regulated by the brain-derived
prothoracicotropic hormone PTTH.51 The overall amino acid
sequences of various insect PTTHs are not very well conserved,
but they are all believed to form homodimers.52 We identified
a gene coding for the Nasonia PTTH (Table 1 and Supporting
Information, Figure S2). Interestingly, this Nasonia PTTH
contains only 5 of the 7 characteristic cysteine residues present
in PTTH sequences from other insects (Figure 8). Recently, it
was found that the Drosophila tyrosine kinase named Torso
was the receptor for Drosophila PTTH.53 In Nasonia, we could
also identify a PTTH receptor ortholog (Table 2).
Pyrokinins. Pyrokinins (PKs) are small neuropeptides char-
acterized by the common C-terminal sequence FXPRLamide.
They can regulate muscle activity, pheromone biosynthesis,

Journal of Proteome Research • Vol. 9, No. 10, 2010 5303

research articles Hauser et al.

Figure 9. Alignment of the CCHamide preprohormones from N. vitripennis (Nv-CCHa1, annotated from WGS AAZX01006578, and
Nv-CCHa2, annotated from WGS AAZX01016958), A. mellifera (Am-CCHa1, XM_625260, and Am-CCHa2, XM_001120020), D.
melanogaster (Dm-CCHa1, NM_001104314, and Dm-CCHa2, NM_142028), A. gambiae (Ag-CCHa, XM_001237549), A. aegypti (Aa-CCHa,
XM_001649897), T. castaneum (Tc-CCHa, annotated from WGS AAJJ01000164), B. mori (Bm-CCHa, NM_001130115), and I. scapularis
(Is-CCHa, XM_002413842, manually corrected). Amino acid residues that are common in at least six sequences are highlighted in gray.
The arrows indicate the cleavage positions of signal peptidase (SP) or prohormone convertase (PC). The immature CCHamide sequences
are boxed. The two cysteine residues, forming a cystine bridge in each CCHamide peptide, are shown in orange; this cystine bridge
is indicated above the sequences.

melanisation, pupariation, diapause, and feeding behavior in
insects.11 There is a subgroup of pyrokinins characterized by
the C-terminal sequence L/MWFGPRLamide, of which the
specific function is largely unknown. In Drosophila, there are
two precursor genes encoding pyrokinins: (i) the capability gene
encoding one pyrokinin-1 (PK-1, which has the C-terminal
sequence LWFGPRLamide) and two capa peptides (character-
ized by the C-terminal sequence FPRVamide, see below);54 and
(ii) the hugin gene encoding one pyrokinin-2 (PK-2, SVPFK-
PRLamide).55 One receptor was identified in Drosophila that
was preferentially activated by PK-1, whereas two other recep-
tors were more specifically activated by PK-2.56,57
Most insects have, like Drosophila, two preprohormone
genes encoding pyrokinins. In Nasonia, however, we only found
one pyrokinin preprohormone gene that encodes three differ-
ent pyrokinins (but no Capa peptides), of which one has the
C-terminal sequence MWFGPRLamide, thus resembling Droso-
phila PK-1 in its structure (Table 1 and Supporting Information,
Figure S2). The proposed structure of this peptide is unusual,
because it is much longer than all other known pyrokinins and
contains a potential cystine bridge.
SIFamide. SIFamide is a highly conserved dodecapeptide
AYRKPPFNGSIFamide that got its name after its C-terminal
three amino acid sequence. It was first isolated from the gray
flesh fly Neobellieria bullata and shortly after also found in
various other insects.58 The physiological functions of SIFamide
are still not fully understood, but targeted cell ablation and RNA
interference experiments in Drosophila suggest that SIFamide
might modulate sexual behavior.59 In Nasonia, we found a
SIFamide precursor that contains one copy of SIFamide that
is identical to the honey bee and Drosophila peptide (Support-
ing Information, Figure S2). Furthermore, we could confirm
the identity of this peptide by MS (Figure 3, Table 1).
sNPF. Short neuropeptides F (sNPF) have the common
C-terminal sequence RLRF/Wamide and are generally occur-
ring in insects, where they control feeding and reproduction.60-62
In Nasonia, there is a sNPF precursor gene encoding a single
amidated sNPF with the sequence SPSLRLRFamide (Supporting
Information, Figure S2). The presence of this peptide and a
less prominent N-terminally extended peptide sequence (AAER-
SPSLRLRFamide) could be verified by MS (Figures 1-3, Table
1).
Tachykinins. Tachykinins stimulate visceral muscles, but
also act as diuretic hormones on Malpighian tubules from
insects.11,63,64 All known insect tachykinin precursors contain
multiple tachykinin peptides. The annotated Nasonia tachy-
kinin precursor gene contains 9 amidated peptides (7 different
variants) having the common C-terminal sequence FXGM/
VRamide characteristic for tachykinins (Table 1 and Supporting
Information, Figure S2). Five of these Nasonia tachykinins
(TK1-5) showed remarkably high signal intensities in mass
spectra from the antennal lobes (Figure 3). No signals from the
remaining two tachykinins (TK-6 and -7), which are both
located in the C-terminal sequence of the preprohormone, were
detectable, which is amazing, because their sequences would
suggest perfect ionization behavior.
B. Neuropeptides with Unknown Receptors in Insects. We
found Nasonia orthologs for 8 insect neuropeptide and protein
hormone precursor genes, where there has not been identified
a receptor in any insect species so far (Table 2, group B). These
are two genes, each coding for a CCHamide peptide; two genes,
each coding for an insulin-like peptide; and one gene each,

5304 Journal of Proteome Research • Vol. 9, No. 10, 2010

Global Analysis of Neuropeptides in Nasonia research articles

Figure 10. Alignment of the RYamide preprohormone from N. vitripennis (Nv-RYa, annotated from WGS AAZX01010442 and
AAZX01007154) with the corresponding preprohormones from A. mellifera (Am-RYa, annotated from WGS AADG05005050), A. gambiae
(Ag-RYa, annotated from EST BM630458 and WGS AAAB01008807), A. aegypti (Aa-RYa, annotated from EST DV310014 and WGS
AAGE02021324), C. pipiens (Cp-RYa, annotated from WGS AAWU01014648 and AAWU01014649), D. melanogaster (Dm-RYa,
NM_001110912, manually corrected), D. grimshawi (Dg-RYa, XM_001987072), D. virilis (Dv-RYa, XM_002049998), D. willistoni (Dw-
RYa, XM_002061063), B. mori (Bm-RYa, annotated from WGS AADK01014080), T. castaneum (Tc-RYa, annotated from WGS
AAJJ01000008), P. humanus (Ph-RYa, XM_002423529), and A. pisum (Ap-RYa, annotated from WGS ABLF02038582). The putative
signal peptides (predicted by SignalP) are highlighted in blue. The immature RYamide peptides are marked with yellow background.
Green background indicates putative cleavage sites for prohormone convertases (PC). Glycine residues that are converted into C-terminal
amide groups in the mature peptides are highlighted with a blue background.

coding for the ion transport peptide (ITP), neuroparsin, and a
partial orcokinin precursor containing 10 orcokinin-like neu-
ropeptides (Table 1 and Supporting Information, Figure S2).
The presence of the orcokinin-like peptides could be confirmed
by MS (Figure 1, Figure 2). In addition, we discovered a novel
neuropeptide gene that codes for 7 peptides, of which 4 have
the C-terminal sequence RYamide and which were named
RYamide peptides (Table 1 and Supporting Information, Figure
S2). All neuropeptides mentioned in this paragraph have been
explained in a very recent review on Drosophila neuropep-
tides,11 except for CCHamide and RYamide peptides. These
neuropeptides are discussed below.
CCHamide. Recently, Roller et al. discovered the novel
neuropeptide CCHamide in B. mori.14 The peptide contains
two highly conserved cysteines and an amidated histidine
residue at the C-terminus. The CCHamide preprohormone is
expressed in several small neurons in the CNS and in endocrine
midgut cells in B. mori larvae, but the biological function of
the peptide is unknown.14 One or two CCHamide precursor
genes are found in all sequenced insect genomes, so far (Figure
9). Also in the Nasonia genome, we identified two paralog
precursor genes (located on different scaffolds), each coding
for a single, slightly different, CCHamide peptide (Table 1;
Figure 9; Supporting Information, Figure S2).
RYamide Peptides. In addition to the above-mentioned 7
neuropeptide and protein hormone genes that are orthologues
of other known insect neuropeptide and protein hormone
genes, we discovered a hitherto unknown insect neuropeptide
gene in Nasonia. This gene codes for a preprohormone that
contains 3 peptides with the C-terminal sequence RFamide and
4 peptides with the C-terminal sequence RYamide (therefore
named RYamide peptides; Figure 10, Figure 11). All peptides
are structurally clearly different from the insect FMRFamide,65
myosuppressin,11 NPF,11 and sNPF60 neuropeptides. Mass
spectrometric analysis of the central nervous system of Nasonia
revealed a mass-match with putative Nasonia RYamide-1

Journal of Proteome Research • Vol. 9, No. 10, 2010 5305

research articles Hauser et al.

Figure 11. Alignment of all mature insect RYamide peptides predicted, so far (see Figure 10). Residues that are conserved in at least
12 mature insect RYamide peptide sequences are highlighted in red. Conserved (similar, but not identical) residues (compared to the
consensus sequence) are highlighted in green. Most insects have one shorter (8-13 residues) and one longer (27-43 residues) variant
of the RYamide peptide. Note that Nasonia is the only insect that contains 7 copies of RYamide peptides.

(Figure 4) in a number of preparations. RYamide-1 is the
shortest of the predicted Nasonia RYamides, which certainly
favors detectability in mass spectra. Nevertheless, signal in-
tensity was not sufficient for fragment analysis.
Homology searches of the other sequenced arthropod ge-
nomes (various Drosophila species, A. aegypti, A. gambiae, C.
pulex, B. mori, T. castaneum, A. mellifera, A. pisum, P. hu-
manus, D. pulex, I. scapularis) revealed orthologs in most of
them (Figure 10). In each arthropod, these novel neuropeptide
genes code for at least two similar peptides with the C-terminal
sequence RYamide or RFamide (Figure 10, Figure 11). For A.
aegypti, we succeeded in the detection and subsequent frag-
mentation of Aedes RYamide-1 (Figure 11, Figure 12). Thus, we
could confirm that the novel RYamide gene is indeed expressed
and that it yields potentially bioactive neuropeptides.
C. Absent Neuropeptides, for Which a Putative Receptor
Could Be Annotated. We were unable to identify a precursor
gene encoding FMRFamide or FMRFamide-like neuropep-
tides.65 However, there is a clear orthologue to the Drosophila
FMRFamide receptor in Nasonia (Table 1, group C).
D. Absent Ligand and Receptor Couples. We could not
identify precursor genes encoding allatostatin-B, capa, the het-
erodimeric glycoprotein hormone subunits alpha2 (GPA2) and
beta5 (GPB5), kinin, proctolin, sex peptide, and sulfakinin (Table
2, group D). Because we also were unable to identify their

5306 Journal of Proteome Research • Vol. 9, No. 10, 2010

Global Analysis of Neuropeptides in Nasonia
Figure 12. CID mass spectrum of a MALDI-TOF mass peak at ([M
+ H]+: 971.5) from the terminal ganglion of Aedes aegypti.
Fragment ions are labeled and confirm the amino acid sequence
of Aedes RYamide-1 (Figure 11), showing that the RYamide gene
is indeed expressed and that its preprohormone is correctly
processed into a neuropeptide.
corresponding receptors (deorphanized in other insects), these
hormonal signaling systems are probably lacking in Nasonia.
E. Absent Ligand/Orphan Receptor Couples. We could not
identify the following neuropeptide or protein hormone pre-
cursors where there is no receptor known so far (orphans):
allatotropin, antidiuretic factors, and the neuropeptide-like
precursor genes NPLP1 to NPLP4 (Table 2, group E). These
absent neuropeptides are discussed in a recent review.11
Discussion and Conclusions
In our current paper, we could identify 51 neuropeptides
and protein hormones from Nasonia (Table 1), which are
encoded by 30 preprohormone genes (Table 2, group A and B;
Supporting Information, Figure S2). One of these prepro-
homone genes is novel and has not been discovered, so far,
in other insects or animal. This gene codes for 7 peptides,
of which 3 peptides have the C-terminal sequence RFamide
research articles
and 4 peptides have the C-terminal RYamide sequence
(Figure 11 and Supporting Information, Figure S2). These
peptides are clearly different from NPF, which is much longer
(36 residues), has the characteristic RVRFamide C-terminal
sequence,andisonlypresentasonecopyinthepreprohormone.11,60
The RYamide peptides are also different from the sNPFs
(short neuropeptides F), which are shorter, variable in size
and have a conserved C-terminus, RLRF/Wamide11,60 or from
the extended insect FMRFamides, which all have a C-
terminus resembling FMRFamide.65 Finally, the insect myo-
suppressins are decapeptides only present as one copy in
the preprohormone and have a rigid structure different from
the RYamide peptides, being X1DVX2HX3FLRFamide (where
X1 is pQ, P, T; X2 is D, G, V; X3 is V, S).11,60 Thus the Nasonia
RYamide peptide gene appears to be a novel neuropeptide
gene different from other genes coding for peptides contain-
ing the RFamide or RYamide C-terminal sequence.
Screening of other arthropods with a sequenced genome
revealed orthologs of the Nasonia RYamide peptide gene
(Figure 10). These orthologs code for preprohormones contain-
ing mainly peptides with the C-terminal sequence RYamide
(Figure 11), supporting our decision to name these peptides
RYamides (and not RFamides). The identification and sequenc-
ing by MS of an Aedes RYamide in the central nervous system
of Aedes aegypti (Figure 12) shows that the RYamide gene is
indeed expressed and that its preprohormone is correctly
processed into a mature neuropeptide. The novel RYamide
genes from insects, therefore, should be regarded as genuine
neuropeptide genes. Nasonia is, so far, the only insect, where
the RYamide gene codes for 7 peptides.
No FMRFamide precursor could be identified, although the
corresponding putative FMRFamide receptor gene is present
in Nasonia (Table 2, group C). This could mean that our
genomics approach fails to detect these neuropeptides, either
because of gaps in the sequenced genome or because the
precursor sequence has diverged too much for homology-based
searches. This last possibility might be the most likely reason,
as FMRFamides are known to have highly variable peptide and
precursor structures in insects.11 However, it can not be
excluded that the putative FMRFamide receptor is, in fact, a
receptor for the RYamide peptides. This interesting possibility
has to be clarified in future studies.
If both the peptide and the receptor genes are lacking in
Nasonia (Table 2, group D), the likelihood that these hormonal

Table 3. Core Set of Neurohormone Precursors Found in Nasonia, Apis, Drosophila, Aedes, Bombyx, Tribolium and the Pea Aphid,
Acyrthosiphon pisum
Peptide Nasonia Apis Drosophila Aedes Bombyx Tribolium Pea Aphid
AKH 1 1 1 1 2 2 1
Alllatostatin C 1 1 1 1 1 1 1
Allatostatin CC 1 1 1 1 1 1 1
Bursicon alpha 1 1 1 1 1 1 1
Bursicon beta 1 1 1 1 1 1 1
CCAP 1 1 1 1 1 1 1
CCHamide 2 2 2 1 1 1 2
DH (Calcitonin-like) 1 1 1 1 1 1 1
DH (CRF-like) 1 1 1 1 1 1 1
EH 1 1 1 5 1 1 3
ETH 1 1 1 1 1 1 1
ILP-B 1 1 5 6 38 2 7
ITP 1 1 1 1 1 1 1
Myosuppressin 1 1 1 1 1 1 1
Pyrokinin 1 1 1 1 1 1 1
RYamide 1 1 1 1 1 1 1
SIFamide 1 1 1 1 2 1 1
sNPF 1 1 1 2 1 1 1
Tachykinin 1 1 1 1 1 1 1
20 20 24 29 58 21 28

Journal of Proteome Research • Vol. 9, No. 10, 2010 5307

research articles Hauser et al.
Table 4. Variable Set of Neurohormone Precursors Found in Nasonia, Apis, Drosophila, Aedes, Bombyx, Tribolium and the Pea
Aphid, Acyrthosiphon pisum
Peptide Nasonia Apis Drosophila Aedes Bombyx Tribolium Pea Aphid
ACP 1 nd nd 1 1 1 nd
ADF-b nd nd nd nd nd 5 nd
Allatostatin A 1 1 1 1 1 nd 1
Allatostatin B nd nd 1 1 1 1 1
Allatotropin nd nd nd 1 1 1 1
Capa nd 1 1 1 1 1 1
Corazonin 1 1 1 1 1 nd nd
FMRFamide nd 1 1 1 1 1 1
GPA2 nd nd 1 1 1 1 1
GPB5 nd nd 1 1 1 1 1
ILP-A nd nd 1 1 nd 1 nd
ILP-C 1 1 1 1 nd 1 3
Inotocin 1 nd nd nd nd 1 nd
Kinin nd nd 1 1 1 nd 1
Neuroparsin 1 1 nd 1 1 1 nd
NPF 1 1 1 1 2 nd 1
NPLP1 nd 1 1 1 1 1 1
NPLP2 nd 1 1 nd nd nd nd
NPLP3 nd 1 1 nd nd nd nd
NPLP4 nd nd 1 nd nd nd nd
Orcokinin 1 1 nd 1 1 nd 1
PDF 1 1 1 1 1 nd nd
Proctolin nd nd 1 nd nd 1 1
PTTH 1 nd 1 1 1 1 nd
Sex peptide nd nd 2 nd nd nd nd
Sulfakinin nd 1 1 1 1 1 nd
10 13 21 19 18 20 15

systems are truly absent in the wasp strongly increases. Two
orthologues of the previously proposed Drosophila allatosta-
tin-B receptor CG3010666 are present in Nasonia, but the
deorphanization of this receptor was questioned recently, as
the Bombyx orthologue of CG30106 did not respond to Bombyx
allatostatins-B.67 Surprisingly, it turned out that the Drosophila
allatostatins-B are potent ligands for another receptor, the
Drosophila sex peptide receptor CG16752,67,68 and it is this
allatostatin-B/sex peptide receptor couple that got lost in
Nasonia.67,69 Like the allatostatin-B/sex peptide receptor, also
the receptors for capa, GPA2 and GPB5, kinin, proctolin, and
sulfakinin are absent in Nasonia.8 We conclude, therefore, that
the signaling systems for allatostatin-B, capa, GPA2 and GPB5,
kinin, proctolin, sex peptide, and sulfakinin do not occur in
Nasonia.
The sets of neuropeptides identified in the wasp N. vitrip-
ennis (this study), the honey bee A. mellifera,9,10 the fruit fly
D. melanogaster,12 the mosquito A. aegypti,13 the silkworm B.
mori,14 the flour beetle T. castaneum15,16 and the pea aphid A.
pisum17,36 can be subdivided into a basal set of 20 neuropeptide
precursors that are present in all these seven arthropod
genomes (Table 3) and into a variable set of 26 precursors that
are not found in all of these genomes (Table 4).
The basal set of peptides shown in Table 3 might contain
the key regulators for common physiological processes such
as development, metabolism, or reproduction. The peptides
listed in Table 4, on the other hand, might regulate other more
specialized processes not required for all insects and, therefore,
can readily get lost during evolution. Nasonia contains, together
with the honey bee, the lowest number of neurohormone
precursor genes in the core set (Table 3). But also in the variable
set (Table 4), this number is by far the lowest in Nasonia (10
compared to 13 in Apis, 15 in the pea aphid, 18 in Bombyx, 19
in Aedes, 20 in Tribolium and 21 in Drosophila), and its
neuropeptide set is remarkably different even from the set
identified in the honey bee, another hymenopteran. In Nasonia,
there is ACP, inotocin, and PTTH, which are neuropeptides not
found in Apis. On the other hand, in the honey bee there is
capa, FMRFamide, three NPLP-like precursors, and sulfakinin;
none of these neuropeptides are present in Nasonia. These
differences might reflect differences in behavior (social vs
parasitic), feeding (herbivores vs carnivores), or habitats of
these two related insect species.
We have previously noticed that insects can readily duplicate
or abandon neuropeptide/GPCR genes.7,8,16,18,46 It is, therefore,
one of the biggest challenges for insect endocrinologists to
understand the basis for this phenomenon and eventually
correlate the presence or absence of neuropeptide signaling
systems with the ecological niche that is occupied by a certain
insect.
Acknowledgment. We thank Professor Jack Werren
(University of Rochester) for sending Nasonia, Professor
Joachim Ruther (University of Regensburg) and Anders Illum
(University of Copenhagen) for help with establishing and
maintaining Nasonia cultures, Louise Lindbæk and Ida
Signe Bohse Larsen (University of Copenhagen) for help
with cDNA cloning of Allatostatin-C and Allatostatin-CC
preprohormones, and the Danish Research Council for
Nature and Universe, German Research Foundation, and
Novo Nordisk Foundation for financial support.
Supporting Information Available: Supplementary
figures and table. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) Saul, G. B.; Kayhart, M. Mutants and linkage in Mormoniella.
Genetics 1956, 41, 930–937.
(2) Beukeboom, L.; Desplan, C. Nasonia. Curr. Biol. 2003, 13, R860.
(3) Werren, J. H. Sex ratio adaptations to local mate competition in a
parasitic wasp. Science 1980, 208, 1157–1159.
(4) Werren, J. H.; Richards, S.; Desjardins, C. A.; Niehuis, O.; et al.
Functional and evolutionary insights from the genomes of three
parasitoid Nasonia species. Science 2010, 327, 343–348.
(5) Grimmelikhuijzen, C. J. P.; Cazzamali, G.; Williamson, M.; Hauser,
F. Perspective. The promise of insect genomics. Pest Manag. Sci.
2007, 63, 413–416.
(6) Weinstock, G. M.; Robinson, G. E.; Gibbs, R. A.; Worley, K. C.;
Evans, J. D.; Maleszka, R.; Robertson, H. M.; Weaver, D. B.; Beye,
M.; Bork, P.; Elsik, C. G.; Hartfelder, K.; Hunt, G. J.; et al. Insights

5308 Journal of Proteome Research • Vol. 9, No. 10, 2010

Global Analysis of Neuropeptides in Nasonia
into social insects from the genome of the honey bee Apis
mellifera. Nature 2006, 443, 931–949.
(7) Hauser, F.; Cazzamali, G.; Williamson, M.; Blenau, W.; Grimme-
likhuijzen, C. J. P. A review of neurohormone GPCRs present in
the fruitfly Drosophila melanogaster and the honey bee Apis
mellifera. Prog. Neurobiol. 2006, 80, 1–19.
(8) Hauser, F.; Cazzamali, G.; Williamson, M.; Park, Y.; Li, B.; Tanaka,
Y.; Predel, R.; Neupert, S.; Schachtner, J.; Verleyen, P.; Grimme-
likhuijzen, C. J. P. A genome-wide inventory of neurohormone
GPCRs in the red flour beetle Tribolium castaneum. Front.
Neuroendocrinol. 2008, 29, 142–165.
(9) Hummon, A. B.; Richmond, T. A.; Verleyen, P.; Baggerman, G.;
Huybrechts, J.; Ewing, M. A.; Vierstraete, E.; Rodriguez-Zas, S. L.;
Schoofs, L.; Robinson, G. E.; Sweedler, J. V. From the genome to
the proteome: uncovering peptides in the Apis brain. Science
2006, 314, 647–649.
(10) Predel, R.; Neupert, S. Social behavior and the evolution of
neuropeptide genes: lessons from the honeybee genome. Bioessays
2007, 29, 416–421.
(11) Nässel, D. R.; Winther, A. M. Drosophila neuropeptides in regula-
tion of physiology and behavior. Prog. Neurobiol. 2010, 92, 42–
104.
(12) Wegener, C.; Gorbashov, A. Molecular evolution of neuropeptides
in the genus Drosophila. Genome Biol. 2008, 9, R131.
(13) Predel, R.; Neupert, S.; Garczynski, S. F.; Crim, J. W.; Brown, M. R.;
Russell, W. K.; Kahnt, J.; Russell, D. H.; Nachman, R. J. Neuropep-
tidomics of the mosquito Aedes aegypti. J. Proteome Res. 2010, 9,
2006–2015.
(14) Roller, L.; Yamanaka, N.; Watanabe, K.; Daubnerová, I.; Zitnan,
D.; Kataoka, H.; Tanaka, Y. The unique evolution of neuropeptide
genes in the silkworm Bombyx mori. Insect Biochem. Mol. Biol.
2008, 38, 1147–1157.
(15) Amare, A.; Sweedler, J. V. Neuropeptide precursors in Tribolium
castaneum. Peptides 2007, 28, 1282–1291.
(16) Li, B.; Predel, R.; Neupert, S.; Hauser, F.; Tanaka, Y.; Cazzamali,
G.; Williamson, M.; Arakane, Y.; Verleyen, P.; Schoofs, L.; Schacht-
ner, J.; Grimmelikhuijzen, C. J. P.; Park, Y. Genomics, transcrip-
tomics, and peptidomics of neuropeptides and protein hormones
in the red flour beetle Tribolium castaneum. Genome Res. 2008,
18, 113–122.
(17) Huybrechts, J.; Bonhomme, J.; Minoli, S.; Prunier-Leterme, N.;
Dombrovsky, A.; Abdel-Latief, M.; Robichon, A.; Veenstra, J. A.;
Tagu, D. Neuropeptides and neurohormone precursors in the pea
aphid, Acyrthosiphon pisum. Insect Mol. Biol. 2010, 19, Suppl. 2,
87–95.
(18) Hansen, K. H.; Stafflinger, E.; Schneider, M.; Hauser, F.; Cazzamali,
G.; Williamson, M.; Kollmann, K.; Schachtner, J.; Grimmelikhuijzen,
C. J. P. Discovery of a novel insect neuropeptide signaling system
closely related to the insect adipokinetic hormone and corazonin
hormonal systems. J. Biol. Chem. 2010, 285, 10736–10747.
(19) Gäde, G.; Hoffmann, K. H.; Spring, J. H. Hormonal regulation in
insects: facts, gaps, and future directions. Physiol. Rev. 1997, 77,
963–1032.
(20) Williamson, M.; Lenz, C.; Winther, Å. M. E.; Nässel, D. R.;
Grimmelikhuijzen, C. J. P. Molecular cloning, genomic organiza-
tion, and expression of a C-type (Manduca sexta-type) allatostatin
preprohormone from Drosophila melanogaster. Biochem. Biophys.
Res. Commun. 2001, 282, 124–130.
(21) Stay, B.; Tobe, S. S. The role of allatostatins in juvenile hormone
synthesis in insects and crustaceans. Annu. Rev. Entomol. 2007,
52, 277–299.
(22) Veenstra, J. A. Allatostatin C and its paralog allatostatin double C:
The arthropod somatostatins. Insect Biochem. Mol. Biol. 2009, 39,
161–170.
(23) Mendive, F. M.; Van Loy, T.; Claeysen, S.; Poels, J.; Williamson,
M.; Hauser, F.; Grimmelikhuijzen, C. J. P.; Vassart, G.; Vanden
Broeck, J. Drosophila molting neurohormone bursicon is a het-
erodimer and the natural agonist of the orphan receptor DLGR2.
FEBS Lett. 2005, 579, 2171–2176.
(24) Luo, C. W.; Dewey, E. M.; Sudo, S.; Ewer, J.; Hsu, S. Y.; Honegger,
H. W.; Hsueh, A. J. Bursicon, the insect cuticle-hardening hormone,
is a heterodimeric cystine knot protein that activates G protein-
coupled receptor LGR2. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
2820–2825.
(25) Luan, H.; Lemon, W. C.; Peabody, N. C.; Pohl, J. B.; Zelensky, P. K.;
Wang, D.; Nitabach, M. N.; Holmes, T. C.; White, B. H. Functional
dissection of a neuronal network required for cuticle tanning and
wing expansion in Drosophila. J. Neurosci. 2006, 26, 573–584.
(26) Peabody, N. C.; Diao, F.; Luan, H.; Wang, H.; Dewey, E.; Honnegger,
H. W.; White, B. H. Bursicon functions within the Drosophila CNS
research articles
to modulate wing expansion behavior, hormone secretion, and
cell death. J. Neurosci. 2008, 28, 14379–14391.
(27) Kimura, K.; Kodama, A.; Hayasaka, Y.; Ohta, T. Activation of the
cAMP/PKA signalling pathway is required for post-ecdysial cell
death in wing epidermal cells of Drosophila melanogaster. Devel-
opment 2004, 131, 1597–1606.
(28) Woodruff, E. A.; Broadie, K.; Honegger, H. W. Two peptide
transmitters co-packaged in a single neurosecretory vesicle. Pep-
tides 2008, 29, 2276–2280.
(29) Gammie, S. C.; Truman, J. W. Eclosion hormone provides a link
between ecdysis-triggering hormone and crustacean cardioactive
peptide in the neuroendocrine cascade that controls ecdysis
behavior. J. Exp. Biol. 1999, 202, 343–352.
(30) Veenstra, J. A. Isolation and structure of corazonin, a cardioactive
peptide from the American cockroach. FEBS Lett. 1989, 250, 231–
234.
(31) Tawfik, A. I.; Tanaka, S.; De Loof, A.; Schoofs, L.; Baggerman, G.;
Waelkens, E.; Derua, R.; Milner, Y.; Yerushalmi, Y.; Pener, M. P.
Identification of the gregarization-associated dark-pigmentotropin
in locusts through an albino mutant. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 7083–7087.
(32) Veenstra, J. A. Does corazonin signal nutritional stress in insects?
Insect Biochem. Mol. Biol. 2009, 39, 755–762.
(33) Boerjan, B.; Verleyen, P.; Huybrechts, J.; Schoofs, L.; De Loof, A.
In search for a common denominator for the diverse functions of
arthropod corazonin: a role in the physiology of stress? Gen. Comp.
Endrocrinol. 2010, 166, 222–233.
(34) Zhao, Y.; Bretz, C. A.; Hawksworth, S. A.; Hirsh, J.; Johnson, E. C.
Corazonin neurons function in sexually dimorphic circuitry that
shape behavioral responses to stress in Drosophila. PLoS One 2010,
5, e9141.
(35) Predel, R.; Neupert, S.; Russell, W. K.; Scheibner, O.; Nachman,
R. J. Corazonin in insects. Peptides 2007, 28, 3–10.
(36) International Aphid Genomics Consortium,Richards, S.; Gibb, R. A.;
Gerardo, N. M.; Moran, N.; et al. Genome sequence of the pea
aphid Acyrthosiphon pisum. PLoS Biol. 2010, 8, e1000313.
(37) Coast, G. M.; Webster, S. G.; Schegg, K. M.; Tobe, S. S.; Schooley,
D. A. The Drosophila melanogaster homologue of an insect
calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit
fly Malpighian tubules. J. Exp. Biol. 2001, 204, 1795–1804.
(38) Cabrero, P.; Radford, J. C.; Broderick, K. E.; Costes, L.; Veenstra,
J. A.; Spana, E. P.; Davies, S. A.; Dow, J. A. The Dh gene of
Drosophila melanogaster encodes a diuretic peptide that acts
through cyclic AMP. J. Exp. Biol. 2002, 205, 3799–3807.
(39) Zitnan, D.; Kim, Y.-J.; Zitnanová, I.; Roller, L.; Adams, M. E.
Complex steroid-peptide-receptor cascade controls insect ecdysis.
Gen. Comp. Endrocrinol. 2007, 153, 88–96.
(40) Morton, D. B.; Simpson, P. J. Cellular signaling in eclosion
hormone action. J. Insect Physiol. 2002, 48, 1–13.
(41) Chang, J. C.; Yang, R. B.; Adams, M. E.; Lu, K. H. Receptor guanylyl
cyclases in Inka cells targeted by eclosion hormone. Proc. Natl.
Acad. Sci. U.S.A. 2009, 106, 13371–13376.
(42) Kono, T.; Nagasawa, H.; Kataoka, H.; Isogai, A.; Fugo, H.; Suzuki,
A. Eclosion hormone of the silkworm Bombyx mori. Expression
in Escherichia coli and location of disulfide bonds. FEBS Lett. 1990,
263, 358–360.
(43) Hull, J. J.; Copley, K. S.; Schegg, K. M.; Quilici, D. R.; Schooley,
D. A.; Welch, W. H. De novo molecular modeling and biophysical
characterization of Manduca sexta eclosion hormone. Biochemistry
2009, 48, 9047–9060.
(44) Zitnan, D.; Kingan, T. G.; Hermesman, J. L.; Adams, M. E.
Identification of ecdysis-triggering hormone from an epitracheal
endocrine system. Science 1996, 271, 88–91.
(45) Zitnan, D.; Zitnanová, I.; Spalovská, I.; Takác, P.; Park, Y.; Adams,
M. E. Conservation of ecdysis-triggering hormone signalling in
insects. J. Exp. Biol. 2003, 206, 1275–1289.
(46) Stafflinger, E.; Hansen, K. K.; Hauser, F.; Schneider, M.; Cazzamali,
G.; Williamson, M.; Grimmelikhuijzen, C. J. P. Cloning and
identification of an oxytocin/vasopressin-like receptor and its
ligand from insects. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3262–
3267.
(47) Wu, Q.; Wen, T.; Lee, G.; Park, J. H.; Cai, H. N.; Shen, P.
Developmental control of foraging and social behavior by the
Drosophila neuropeptide Y-like system. Neuron 2003, 39, 147–161.
(48) Riehm, J. P.; Rao, K. R. Structure-activity relationships of a
pigment-dispersing crustacean neurohormone. Peptides 1982, 3,
643–647.
(49) Rao, K. R.; Mohrherr, C. J.; Riehm, J. P.; Zahnow, C. A.; Norton, S.;
Johnson, L.; Tarr, G. E. Primary structure of an analog of crustacean
pigment-dispersing hormone from the lubber grasshopper Roma-
lea microptera. J. Biol. Chem. 1987, 262, 2672–2675.
Journal of Proteome Research • Vol. 9, No. 10, 2010 5309
research articles
(50) Renn, S. C.; Park, J. H.; Rosbash, M.; Hall, J. C.; Taghert, P. H. A
pdf neuropeptide gene mutation and ablation of PDF neurons
each cause severe abnormalities of behavioral circadian rhytms
in Drosophila. Cell 1999, 99, 791–802.
(51) Gilbert, L. I.; Rybczynski, R.; Warren, J. T. Control and biochemical
nature of the ecdysteroidogenic pathway. Annu. Rev. Entomol.
2002, 47, 883–916.
(52) Ishizaki, H.; Suzuki, A. The brain secretory peptides that control
moulting and metamorphosis of the silkmoth, Bombyx mori. Int.
J. Dev. Biol. 1994, 38 (2), 301–310.
(53) Rewitz, K. F.; Yamanaka, N.; Gilbert, L. I.; O’Connor, M. B. The
insect neuropeptide PTTH activates receptor tyrosine kinase torso
to initiate metamorphosis. Science 2009, 326, 1403–1405.
(54) Kean, L.; Cazenave, W.; Costes, L.; Broderick, K. E.; Graham, S.;
Pollock, V. P.; Davies, S. A.; Veenstra, J. A.; Dow, J. A. Two
nitridergic peptides are encoded by the gene capability in Droso-
phila melanogaster. Am. J. Physiol. Regul. Integr. Comp. Physiol.
2002, 282, R1297–R1307.
(55) Meng, X.; Wahlström, G.; Immonen, T.; Kolmer, M.; Tirronen, M.;
Predel, R.; Kalkkinen, N.; Heino, T. I.; Sariola, H.; Roos, C. The
Drosophila hugin gene codes for myostimulatory and ecdysis-
modifying neuropeptides. Mech. Dev. 2002, 117, 5–13.
(56) Cazzamali, G.; Torp, M.; Hauser, F.; Williamson, M.; Grimme-
likhuijzen, C. J. P. The Drosophila gene CG9918 codes for a
pyrokinin-1 receptor. Biochem. Biophys. Res. Commun. 2005, 335,
14–19.
(57) Rosenkilde, C.; Cazzamali, G.; Williamson, M.; Hauser, F.; Søn-
dergaard, L.; DeLotto, R.; Grimmelikhuijzen, C. J. P. Molecular
cloning, functional expression, and gene silencing of two Droso-
phila receptors for the Drosophila neuropeptide pyrokinin-2.
Biochem. Biophys. Res. Commun. 2003, 309, 485–494.
(58) Verleyen, P.; Huybrechts, J.; Baggerman, G.; Van Lommel, A.; De
Loof, A.; Schoofs, L. SIFamide is a highly conserved neuropeptide:
a comparative study in different insect species. Biochem. Biophys.
Res. Commun. 2004, 320, 334–341.
(59) Terhzaz, S.; Rosay, P.; Goodwin, S. F.; Veenstra, J. A. The neu-
ropeptide SIFamide modulates sexual behavior in Drosophila.
Biochem. Biophys. Res. Commun. 2007, 352, 305–310.
5310 Journal of Proteome Research • Vol. 9, No. 10, 2010
Hauser et al.
(60) Vanden Broeck, J. Neuropeptides and their precursors in the
fruitfly, Drosophila melanogaster. Peptides 2001, 22, 241–254.
(61) Mertens, I.; Meeusen, T.; Huybrechts, R.; De Loof, A.; Schoofs, L.
Characterization of the short neuropeptide F receptor from
Drosophila melanogaster. Biochem. Biophys. Res. Commun. 2002,
297, 1140–1148.
(62) Lee, K. S.; You, K. H.; Choo, J. K.; Han, Y. M.; Yu, K. Drosophila
short neuropeptide F regulates food intake and body size. J. Biol.
Chem. 2004, 279, 50781–50789.
(63) Siviter, R. J.; Coast, G. M.; Winther, Å. M. E.; Nachman, R. J.; Taylor,
C. A. M.; Shirras, A. D.; Coates, D.; Isaac, R. E.; Nässel, D. R.
Expression and functional characterization of a Drosophila neu-
ropeptide precursor with homology to mammalian preprotachy-
kinin A. J. Biol. Chem. 2000, 275, 23273–23280.
(64) Van Loy, T.; Vandersmissen, H. P.; Poels, J.; Van Hiel, M. B.;
Verlinden, H.; Vanden Broeck, J. Tachykinin-related peptides and
their receptors in invertebrates: a current view. Peptides 2010, 31,
520–524.
(65) Nambu, J. R.; Murphy-Erdosh, C.; Andrews, P. C.; Feistner, G. J.;
Scheller, R. H. Isolation and characterization of a Drosophila
neuropeptide gene. Neuron 1988, 1, 55–61.
(66) Johnson, E. C.; Bohn, L. M.; Barak, L. S.; Birse, R. T.; Nässel, D. R.;
Caron, M. G.; Taghert, P. H. Identification of Drosophila neu-
ropeptide receptors by G protein-coupled receptors-beta-arrestin2
interactions. J. Biol. Chem. 2003, 278, 52172–52178.
(67) Yamanaka, N.; Hua, Y. J.; Roller, L.; Spalovská-Valachová, I.;
Mizoguchi, A; Kataoka, H.; Tanaka, Y. Bombyx prothoracicostatic
peptides activate the sex peptide receptor to regulate ecdysteroid
biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2060–2065.
(68) Yapici, N.; Kim, Y. J.; Ribeiro, C.; Dickson, B. J. A receptor that
mediates the post-mating switch in Drosophila reproductive
behaviour. Nature 2008, 451, 33–37.
(69) Kim, Y. J.; Bartalska, K.; Audsley, N.; Yamanaka, N.; Yapici, N.; Lee,
J. Y.; Kim, Y. C.; Markovic, M.; Isaac, E.; Tanaka, Y.; Dickson, B. J.
MIPs are ancestral ligands for the sex peptide receptor. Proc. Natl.
Acad. Sci. U.S.A. 2010, 107, 6520–6525.
PR100570J