TMP BD5 E

ARTICLE
pubs.acs.org/jpr
Genomics, Transcriptomics, and Peptidomics of Daphnia pulex

Neuropeptides and Protein Hormones
Heinrich Dircksen,*,† Susanne Neupert,‡ Reinhard Predel,‡ Peter Verleyen,§ Jurgen Huybrechts,§
Johannes Strauss,† Frank Hauser,|| Elisabeth Stafflinger,|| Martina Schneider,|| Kevin Pauwels,§
Liliane Schoofs,§ and Cornelis J. P. Grimmelikhuijzen||
†
Department of Zoology, Stockholm University, Sweden
‡
Institute of Zoology, University of Cologne, Cologne, Germany
§
Department of Biology, K.U. Leuven, Belgium
)
Center for Functional and Comparative Insect Genomics, Department of Biology, University of Copenhagen, Denmark
bS Supporting Information
ABSTRACT: We report 43 novel genes in the water flea Daphnia pulex encoding
73 predicted neuropeptide and protein hormones as partly confirmed by RT-
PCR. MALDI-TOF mass spectrometry identified 40 neuropeptides by mass
matches and 30 neuropeptides by fragmentation sequencing. Single genes
encode adipokinetic hormone, allatostatin-A, allatostatin-B, allatotropin, Ala7-
CCAP, CCHamide, Arg7-corazonin, DENamides, CRF-like (DH52) and calci-
tonin-like (DH31) diuretic hormones, two ecdysis-triggering hormones, two
FIRFamides, one insulin, two alternative splice forms of ion transport peptide
(ITP), myosuppressin, neuroparsin, two neuropeptide-F splice forms, three
periviscerokinins (but no pyrokinins), pigment dispersing hormone, proctolin,
Met4-proctolin, short neuropeptide-F, three RYamides, SIFamide, two sulfaki-
nins, and three tachykinins. There are two genes for a preprohormone containing
orcomyotropin-like peptides and orcokinins, two genes for N-terminally elongated ITPs, two genes (clustered) for eclosion
hormones, two genes (clustered) for bursicons alpha, beta, and two genes (clustered) for glycoproteins GPA2, GPB5, three genes
for different allatostatins-C (two of them clustered) and three genes for IGF-related peptides. Detailed comparisons of genes or their
products with those from insects and decapod crustaceans revealed that the D. pulex peptides are often closer related to their insect
than to their decapod crustacean homologues, confirming that branchiopods, to which Daphnia belongs, are the ancestor group of insects.
KEYWORDS: Daphnia pulex, crustacean, neuropeptide, protein hormone, preprohormone, genomics, transcriptomics, bioinformatics,
MALDI-TOF mass spectrometry, RT-PCR
’ INTRODUCTION (RPCH1), later found to occur in all decapods, before its functional
Neuropeptides and peptide hormones are the most diverse antagonist, the pigment dispersing hormone (PDH), was identified.
group of signaling molecules evolved to regulate most physiolo- However, the major products of the X-organ sinus gland (XOSG)
gical processes of animals. In arthropods, especially in insects and neurosecretory system are, apart from RPCH, large peptides of the
crustaceans, peptide hormones have been initially detected by so-called crustacean hyperglycemic hormone (CHH), molt-inhibit-
endocrinological ablation and reconstitution experiments to ing hormone (MIH), and gonad/vitellogenesis inhibiting hormones
investigate endocrine control of a wealth of vital functions, (GIH/VIH) neuropeptide families.2 Members of the CHH peptide
family exert at least eight well-established stimulatory and inhibitory
before the same or similar peptides were also found to act as
actions related to growth and metabolism, whereas the MIHs and
neuromodulators in the central nervous system. In decapod
GIH/VIHs appear to be restricted to the inhibition of ecdyster-
crustaceans, the first neuropeptides with neurohormonal func-
oidogenesis of the Y-organ and of gonad development, respectively.3
tions have been discovered as chromatophorotropic factors In decapods, neurons of the eyestalk medulla are known to produce
eliciting color change in shrimp in vivo-bioassays or by eyestalk PDHs, some of which are released via the sinus gland.4
ablation experiments as factors being “diabetogenic” and respon- Other peptides were discovered in the pericardial organs,
sible for growth inhibition. At least four different types of such which are large neurohemal organs consisting of nerve endings
hormonal peptides had already been identified up to the 1990s. next to the heart of decapod crustaceans. These peptides
They originate from the so-called X-organ or from other eyestalk are affecting heart rate and volume output when tested in
ganglia and are released via the neurohemal sinus gland into the
hemolymph. The first crustacean peptide to be fully sequenced Received: March 29, 2011
was the nonapeptide red pigment concentrating hormone Published: August 10, 2011
r 2011 American Chemical Society 4478 dx.doi.org/10.1021/pr200284e | J. Proteome Res. 2011, 10, 4478–4504
Journal of Proteome Research ARTICLE
semi-isolated preparations and were identified as proctolin,5 Tris-base (0.61 g/L). Brain-optic ganglia complexes were collected
crustacean cardioactive peptide (CCAP),6 and molluscan on ice in Eppendorf caps containing 50 μL of 2 M aqueous acetic
FMRFamide-related peptides. Several of these peptides were acid solution. The samples were sonicated (on ice) three times for
later also found to be myoactive on certain visceral (e.g., hindgut) 1 min. After spinning down debris, the extracts were concentrated
and skeletal muscles.7 Other myoactive peptides such as the and desalted using ZipTipC18 (Millipore, 15 μm). The ZipTipC18
orcokinins and orcomyotropin8,9 were discovered in crayfish was pre-equilibrated for sample binding using 0.1% aqueous TFA
hindgut bioassays. Additional peptides were detected in com- containing 50% CH3CN and subsequently washed with 0.1%
parative studies by use of separation techniques such as high aqueous TFA. The samples were loaded onto the ZipTipC18 and
performance liquid chromatography (HPLC) combined with washed with 0.1% aqueous TFA. Then, samples were eluted in 1 μL
enzyme-immunoassays, for example, the large peptide family of acetonitrile/water/formic acid (70:29.9:0.1; v/v/v) directly onto
crustacean allatostatins.1012 However, with the enormous tech- the MALDI target and mixed with 0.5 μL saturated alpha-cyano-4-
nical improvements and increased sensitivity of mass spectro- hydroxycinnamic acid in ethanol/CH3CN/TFA (50:49.9:0.1; v/v/v),
metry (MS) techniques including fragmentation capabilities air-dried, and analyzed. MALDI-TOF-MS was performed on a
(MS/MS), another peak of peptide discoveries recently occurred Reflex IV instrument (Bruker Daltonik, Bremen, Germany),
for several decapod species, mainly crabs, crayfish, and lobsters, equipped with an N2 laser (337 nm) and pulsed ion extraction
so that we now know more than 200 different crustacean accessory. The instrument was operated in the positive ion,
peptides.13 reflectron mode and calibrated using a standard peptide mixture
Among the first peptide gene structures available were those containing angiotensin II (1045.54 Da), angiotensin I (1295.68
encoding classical CHHs and MIHs. As a complication, however, Da), substance P (1346.73 Da), bombesin (1618.82 Da), ACTH
decapod CHHs and even MIHs are not derived from single genes Clip 117 (2092.08 Da) and ACTH Clip 1839 (2464.19 Da)
only but from distinct clusters of 10 or more genes in tandem (Bruker Daltonik). The instrument settings were as follows: ion
arrangement on the same chromosome as has been worked out in source 1, 25.00 kV; ion source 2, 20.35 kV; pulsed ion extraction set
great detail for some shrimp species.14 Genome sequencing, at 200 ns; lens voltage, 12.30 kV; and reflector voltage, 28.70 kV.
expressed sequence tag (EST) library collections, and the avail- Matrix deflection was set at 500 Da and the laser energy was adjusted
ability of more sophisticated bioinformatics techniques for gene to just above desorption/ionization threshold. Spectra were re-
discovery and annotation allowed reading precursor and peptide corded in the reflectron mode within a mass range from m/z 500 to
structures from the (re)constructed genes. Thus, the finishing of m/z 3000 and are the results of ca. 100 shots.
the genome of Daphnia pulex prompted us as members of the B). Direct Tissue Profiling. Single specimens of D. pulex
Daphnia Genome Consortium (DGC) annotation team15 to were pinned with microneedles and submerged in an insect saline
use the available genomic information not only to further annotate (pH 7.25) of the following composition: NaCl (7.50 g/L), KCl
several peptide genes but also to confirm peptide hormone (0.20 g/L), CaCl2 (0.20 g/L) and NaHCO3 (0.10 g/L). The
precursors and to identify their derived neuropeptide(s) in the body cavities were opened with fine forceps and ultrafine scissors,
central nervous system of this species. Thus, this report describes the brains dissected and subsequently cut into smaller pieces,
for the first time the global analysis of neuropeptide genes and which were transferred with a glass capillary into a drop of
their products in a crustacean. We have in all cases newly distilled water on the sample plate for MALDI-TOF mass
annotated or thoroughly revised the computer-generated pep- spectrometry. The water was removed using the same glass
tide-gene structures, partly confirmed their cDNAs by RT-PCR capillary. The tissues were air-dried and covered with approxi-
and confirmed more than half of the predicted neuropeptides by mately 50 nL (depending on the sample size) of matrix solution
mass spectrometry. Our analysis revealed that Daphnia neuro- (saturated alpha-cyano-4-hydroxycinnamic acid dissolved in
peptides were often more similar to insect than to decapod methanol/water 1:1) over a period of about 5 s using a Nanoliter
crustacean neuropeptides, supporting the view that branchio- injector (World Precision Instruments, Berlin, Germany). Each
pods, for example, tadpole shrimps and water fleas, to which preparation was air-dried again and covered with distilled water
Daphnia pulex belongs, are the ancestor group of insects.16 for a few seconds, which was finally removed with cellulose paper
to reduce salt contents. MALDI-TOF analyses were performed
’ MATERIALS AND METHODS on an ABI 4800 Proteomics Analyzer (Applied Biosystems,
Adult parthenogenetic females of water fleas Daphnia pulex Framingham, MA). To determine the parent masses, the instru-
(Leydig 1860) (strain Livpu01, Dept. Biology, Leuven University, ment was operated in reflectron mode. Tandem MS experiments
Leuven, Belgium; clone originally hatched from resting eggs from a were performed in gas-off mode. The number of laser shots used
natural population at Brown Moss, North Shropshire, U.K.) were to obtain a spectrum varied from 800 to 4000, depending on
used for mass spectrometry and de novo-sequencing experiments. signal quality, and a mass range from m/z 600 to m/z 5000 was
Animals were fed ad libitum (food concentrations: 150 000 cells chosen. The fragmentation patterns were analyzed by use of the
per mL corresponding to 1.1 mg cells per L) green algae Scenedesmus Data ExplorerT software 4.3 package to confirm the sequence of
obliquus (K€utzing 1833:609) reared under laboratory conditions, the peptide.
(20 ( 2 °C; 14 h light/10 h dark photoperiod) in dechlorinated tap
Bioinformatics
water (30 animals in 3 L jars). Animals were raised until they
As bioinformatics tools BLAST searches were usually per-
reached second or third adult instar before extracting peptides.
formed applying the tblastn or blastp algorithms at NCBI, the
Sample Preparation and Mass Spectrometry Joint Genome Institute (JGI), or the www.wfleabase.org bioin-
A). Extraction. Batches of up to 50 specimens of D. pulex were formatics Web sites. Alignments were performed with the
dissected in a modified Ringer solution17 (pH 7.5) of the following CLUSTALW algorithm embedded within the BioEdit program
composition: NaCl (12.0 g/L), KCl (0.4 g/L), CaCl2 2H2O version 7.0.5.3 or for small sequences fine-tuned by hand or put
(2.25 g/L), MgCl2 6H2O (0.5 g/L), maleic acid (0.58 g/L), into in the Genedoc program version 2.7.0. The statistics output
4479 dx.doi.org/10.1021/pr200284e |J. Proteome Res. 2011, 10, 4478–4504
Table 1. Alphabetical List of Mature Peptides and Protein Hormones of Daphnia pulex
mass annotated/PCR-
peptide name peptide sequence(s) [M + H]+ match MS/MS confirmed
Adipokinetic hormone pQVNFSTSWa 950.44 + +/nd

Allatostatin A1 SFGGNPTGDPNLNIYSFGLa 1968.94 +/+
Allatostatin A2 TSRSYSINPYSFGLa 1590.79 + + +/+
Allatostatin A3 GGNAKSYPQQIPYSFGLa 1825.92 +/+
Allatostatin A4 NPTKYNFGLa 1052.55 + + +/+
Allatostatin A5 PDRFGFGLa 907.48 + + +/+
Allatostatin A6 LPVYNFGLa 921.52 + +a +/+
Allatostatin B1 NNWNRMQGMWa 1335.58 + +a +/nd
Allatostatin B2 AWSDLSQQGWa 1176.54 +/nd
Allatostatin B3 SWTQLHGVWa 1112.56 + +a +/nd
Allatostatin B4 RWDQLHGAWa 1167.58 + + +/nd
Allatostatin B5 SGWNKMQGVWa 1191.57 + + +/nd
Allatostatin B6 GWNQLQGVWa 1086.54 +/nd
Allatostatin B7 NWNNLRGAWa 1129.56 + + +/nd
Allatostatin B8 ESGWNNLKGLWa 1302.65 +/nd
Allatostatin C1 SYWKQCAFNAVSCFa 1650.72 +/nd
Allatostatin C2 GQSSQRVFWRCYFNAVSCF 2283.02 +/nd
Allatostatin C3 SKQLRYHHCYFNPISCF 2140.98 +/nd
Allatotropin GFKTVGLATARGFa 1323.75 + + +/+
Bursicon-alpha [Dappu1:306405] nd +/nd
Bursicon-beta [Dappu1:221518] nd +/nd
CCHamide NCNKYGNACFGAHa 1395.56 +/nd
Corazonin pQTFQYSRGWTNa 1369.63 + + +/nd
Crustacean cardioactive peptide PFCNAFAGCa 926.36 +/nd
DENamide 1 KCHFDENa 891.38 +/nd
DENamide 2 FMGLSHFDENa 1195.52 +/nd
DENamide 3 FKGLSHFDENa 1192.57 +/nd
DENamide 4 FKGLSHFGENa 1134.57 +/nd
Diuretic hormone-31 GVDFGLGRGYSGSQAAKHLMGLAAANYAIGPa 3048.55 + + +/+
Diuretic hormone-52 [Dappu1:304695] nd +/nd
Ecdysis-triggering hormone 1 DPSPEPFNPNYNRFRQKIPRIa 2584.35 +/nd
Ecdysis-triggering hormone 2 GEGIIAEYMNSESFPHEGSLSNFFLKASKAVPRLa 3724.88 +/nd
Eclosion hormone [Dappu1:240158] nd +/nd
Eclosion hormone long [Dappu1:442999] nd +/nd
FIRFamide 1 SALNKNFIRFa 1208.69 + + +/nd
FIRFamide 2 DEERSFHPARPSRSLRSNFIRFa 2703.39 + +/nd
FIRFamide 21422 SLRSNFIRFa 1138.65 + +/nd
Glycoprotein A2 [Dappu1:212447] nd +/nd
Glycoprotein B5 [Dappu1:319430] nd +/nd
Inotocin CFITNCPPGa 948.40 +/+
Insulin-related peptide-1 [Dappu1:226060] nd +/nd
Insulin-related peptide-2 B-chain NARYCGSYLADALRMACS 1964.88 + +/+
Insulin-related peptide-2 A-chain GVHDECCVKGCTFKELTSYCTRPN 2689.19 +/+
Ion transport peptides (ITP)
ITP (short splice form of ITP) [Dappu1:322492] nd +/+
ITPL (long splice form of ITP) [Dappu1:442877] nd +/+
ITPLN (long, N-term. elongated) [hxAUG26res61g65t1][Dappu1:57088] nd +/nd
ITPN (short, N-term. elongated) [Dappu1:307029] nd +/nd
Myosuppressin [pQ] pQDVDHVFLRFa 1257.64 + + +/+
Myosuppressin [Q] QDVDHVFLRFa 1274.66 + + +/+

Table 1. Continued
mass annotated/PCR-
peptide name peptide sequence(s) [M + H]+ match MS/MS confirmed
Neuroparsin [Dappu1:63582] nd +/+
Neuropeptide F (splice form 1) DGGDVMSGGEGGEMTAMADAIKYLQGLDKVYGQAARPRFa 4060.93 + +/+
Neuropeptide F (splice form 2) [Dappu1:224782] nd +/+
Orcokinin 1 NLDEIDRSNFGTFA 1598.75 + +/nd
Orcokinin 2 NLDEIDRSDFGRFV 1682.81 + +/nd
Orcokinin 3 NLDEIDRSDFSRFV 1712.82 +/nd
Orcomyotropin-like peptide 1 LDSLTGLGFGSQ 1194.60 +/nd
Orcomyotropin-like peptide 2 GLDSLSGASFGIE 1252.61 +/nd
Orcomyotropin-like peptide 3 FDSLTGLGINSQ 1251.62 +/nd
Pigment dispersing hormone NSELINSLLGLPRFMKVVa 2029.16 + + +/+
Periviscerokinin 1 APPQILKSQSLIPFPRVa 1890.12 + + +/nd
Periviscerokinin 2 HLIPFPRVa 977.60 + + +/nd
Periviscerokinin 3 [pQ] pQNLIPFPRVa 1065.62 + + +/nd
Periviscerokinin 3 [Q] QNLIPFPRVa 1082.65 + +/nd
Proctolin RYLPT 649.37 + + +/nd
Met4-Proctolin RYLMT 683.36 + +a +/nd
RYamide 1 pQTFFTNGRYa 1115.53 + + +/nd
RYamide 2 SEVRSRVASRSADERFFGGPRFa 2512.29 +/nd
RYamide 3 SGNGGIVLGNSELDARNPERFFIGSRYa 2924.48 + +/nd
RYamide 31727 NPERFFIGSRYa 1384.71 + +a +/nd
short neuropeptide F SDRSPSLRLRFa 1332.75 + + +/nd
SIFamide TRKLPFNGSIFa 1278.73 + +/nd
Sulfakinin 1 [pQ] pQPDDYGHMRYa 1263.52 + + +/+
Sulfakinin 1 [Q] QPDDYGHMRYa 1280.55 + + +/+
Sulfakinin 2 DFDDYGHMRFa 1301.54 + + +/+
Tachykinin-related peptide 1 TPNSRAFLGMRa 1248.66 + +a +/+
Tachykinin-related peptide 2 KMHGEKFLGMRa 1332.70 + +a +/+
Tachykinin-related peptide 3 APSSNSFMGMRa 1183.54 + +a +/+
a
Contaminated with fragments of other neuropeptides; -a, C-terminal -amide; nd, not determined; for longer peptide sequences the protein identifier
(see Suppl. Table S1, Supporting Information) is provided in brackets.
of Genedoc served for quantifications of peptide similarities, Veenstra, 2000,19 or using the web-based NeuroPred program
by giving either the percent amino acid identity plot (aai) or (http://neuroproteomics.scs.illinois.edu/cgi-bin/neuropred.py).20
the number of residues whose juxtaposition yields a greater
than zero score in the scoring table (scr), i.e. a measure in Reverse Transcription-PCR and Product Sequencing
percent of similar or conservative aa-residues and/or aa- Batches of 50 entire D. pulex individuals were snap-frozen and
substitutions. All shadings in following figures represent pulverized using mortar and pestle under liquid nitrogen and
identities (black) or highly similar aas (gray). For nucleotide processed for total RNA extraction by the Trizol method according
sequence analyses of mRNAs and genes Vector NTI v.9.0.0 to the manufacturer’s instructions (Invitrogen). For generation of
(Informax/Invitrogen) was applied. More detailed analyses cDNA used for 50 - and 30 -rapid amplification of cDNA ends
(RACE), the mRNAs were isolated using the GenElute mRNA
and novel annotations of genes, their exon-intron boundaries
miniprep kit (Sigma-Aldrich). cDNA was then prepared from ca.
were performed usually with aid of Fgenesh-M gene finding
110 μg of isolated mRNA using the SMARTer PCR cDNA
tools (Softberry Inc., Mount Kisco, NY; http://linux1.soft-
Synthesis kit (Clontech-Takara, BD Biosciences). In addition, total
berry.com/berry.phtml?topic=fgenesh-m&group=programs& RNA preparations also served for regular reverse transcription-
subgroup=gfind) applying templates from insects (e.g., Tribo- polymerase chain reactions (RT-PCRs). The RTs were carried out
lium castaneum) and/or GeneSplicer (http://www.cbcb.umd. using either Superscript II reverse transcriptase (Invitrogen) and an
edu/software/GeneSplicer/gene_spl.shtml). Signal peptides oligo-dT-adapter (50 -GCTGTCAACGATACGCTACGTAACG-
have been determined with aid of the SignalP 3.0 server18 GCATGACAGTGT18V-30 ), or Moloney murine leukemia virus
(http://www.cbs.dtu.dk/services/SignalP/) and its evaluating (MMLV)-reverse transcriptase (Promega) and an oligo-dT anchor
algorithms based on neural networks (NN) and hidden Markov primer (50 -GACCACGCGTATCGATGTCGACT16V-30 ) for 50
models (HMM). The sulfinator predictor (http://expasy.org/ min for both at 42 °C followed by an inactivation for 15 min at 72 °C
tools/sulfinator/) served for the analysis of post-translationally and cooling on ice.
sulphated tyrosines in predicted peptides. Putative cleavage sites For RT-PCRs, thermostable DNA polymerase (GoTaq green
in preprohormones have been analyzed according to the rules of kit, Promega) or the Advantage2 PCR kit (Clontech-Takara) were
Figure 1. MALDI-TOF mass spectra from direct profiling of a D. pulex brain. (A) Altogether, 37 peptides and 4 precursor-related peptides (PRPs)
originating from 17 different neuropeptide families were assigned; inset (arrow; stippled rectangle) shows enlarged FIRFamide 2 peak. (BD) Details
from the boxed ranges in the upper complete mass spectrum. ASTA, allatostatin A-type; ASTB, allatostatin B-type; AT, allatotropin, CPON, carboxy-
terminal peptide of NPY; DH31, diuretic hormone 31; IRP-2, insulin-related peptide 2; MS, myosuppressin; NPF, neuropeptide F; RYa, RYamide; SIFa,
SIFamide; SK, sulfakinin; sNPF, short neuropeptide F; OK, orcokinin; TKRP, tachykinin-related peptide.
used both following the manufacturers’ instructions. Gene-specific for 0.52 min and finally for 69 min depending upon product
primers were created from genome sequences and synthesized length.
commercially (MWG, Munich, Germany, Suppl. Table S2, Sup- PCR products obtained with gene-specific primers are listed in
porting Information). For PCRs, touch-down conditions were used Supplementary Table S2 (Supporting Information). In some
as follows: 95 °C for 1 min, 4 cycles of 95 °C for 20 s, 72 °C for cases, they were purified from excised agarose gel pieces (QIAEX
1 min, 5 cycles of 95 °C for 30 s, 70 °C for 1 min, and 36 cycles of II gel purification kit, Qiagen) and directly sequenced commer-
95 °C for 1 min, annealing at 5860 °C depending on the cially (LGC Genomics, Berlin, Germany). For sequence and
respective primers for 1 min, elongation at 68 °C for 1 min, and bioinformatics analyses Vector NTI v.9.0.0 (Informax/In-
finally for 3 min. For PCRs other than RACE-PCRs, the touch- vitrogen) or BioEdit v.7.0.5.3 was applied.
down conditions were modified: 94 °C for 2.5 min, 5 cycles of 94 °C
for 0.5 min, 72 °C for 1.5 min, 5 cycles of 94 °C for 0.5 min, ’ RESULTS
70 °C for 1.5 min, 26 cycles of 94 °C 1 min, annealing at 5665 °C An overview of our results is given in Table 1. We have
depending upon the primers for 0.51 min, elongation at 72 °C identified and annotated 43 D. pulex genes that code for 73
Figure 2. Six novel Dappu allatostatin A-type peptides. (A) None of the
DappuASTA peptides containing a typical Y/FXFGLamide-motif is
identical to any known decapod crustacean or insect ASTA. Asterisks
indicate peptides identified by mass spectrometry. (B) MALDI-TOF/
TOF fragmentation spectrum (direct profiling) of DappuASTA5 at m/z
907.48 from a brain preparation with identified b- and y-fragment series
(P, immonium ion of proline).
predicted neuropeptides from 45 precursors (Table 1; Suppl.

Table S1, Supporting Information). The final annotations were
all checked by gene prediction programs, and many neuropep-
tide gene structures were supported by expressed sequence tags
(ESTs) and/or confirmed by RT-PCR and RACE methods
(Suppl. Table S2, Supporting Information). MALDI-TOF mass Figure 3. Identified Dappu allatostatin B peptides compared with those
spectrometry applied to water flea brain tissue established mass from other arthropods. (A) Seven of the eight DappuASTB-peptides
matches for 40 neuropeptides and seven precursor-related pep- show the typical W(X6)W-amide structure (*identified by direct profil-
tides (PRPs), of which 30 neuropeptides and four PRPs were ing fragmentation analysis). (B) MALDI-TOF/TOF fragmentation
sequenced by MS/MS (Figure 1, Suppl. Tables S1 and S3, spectrum of DappuASTB7 with labeled y- and b-fragments and im-
Supporting Information). These peptides and protein hormones monium ions. (C) Alignments of DappuASTBs (underlined) with the
are dealt with alphabetically in the following. closest related crustacean (dark blue) and insect (green) ASTBs. Only
DappuASTB5 shows higher aa-identities with lobster ASTB than with
Adipokinetic Hormone (AKH)/RPCH-Related Peptide cockroach (Periplaneta americana PeramASTB152) and fly ASTBs, but
not with crab Cancer borealis CanboASTB1. DappuASTB6,-7,-8 are
In decapod crustaceans, RPCH is highly conserved21 but
closer related to insect ASTBs, DappuASTB8 being particularly close to
structures of crustacean RPCH genes were hitherto known only DromeASTB4. (Crustacean ASTBs from: lobster Homarus americanus
for the crab Callinectes sapidus.22 Decapod RPCH is closely ASTB, crab Cancer borealis CanboASTB1 (identical to Carcinus maenas
related to a large number of insect AKHs.23 ASTB13); insect ASTBs from: Drosophila melanogaster DromeASTB4,153
The D. pulex genome contains a single gene similar in cricket Gryllus bimaculatus GrybiASTB6 Q5QRY7).
structure to the decapod RPCH gene in C. sapidus22 and insect
AKH-genes (Suppl. Figure S1A, Supporting Information). The
derived precursor is similar in structure to those of decapod 6 and 7, but shares up to two more identities with some AKHs
RPCH precursors and some insect AKH precursors.23 It con- from hemimetabolous insects such as GrybiAKH and Anai-
sists of a 21aas SP, an AKH/RPCH-like octapeptide with a mAKH (Val2Thr6 or Val2Ser7, respectively). Other closely
C-terminal glycine as putative amide-donor adjacent to a typical related AKHs from insects with maximally two exchanges (as
dibasic KR-processing site, and a precursor related peptide e.g. in bug CorpuAKH, locust SchniAKHII, and beetle Tri-
(PRP). We confirmed the proposed octapeptide as pQVN- caAKH1, among further AKH-isoforms with intermediate
FSTSWamide by mass spectrometry (Table 1). As this peptide characters) share the Thr6 or the Ser7 but are with a Leu2,
is much more similar to AKHs from insects than to decapod Pro6 or Gly7 similar to RPCH (Suppl. Figure S1B, Supporting
RPCH, it is adequate to call it DappuAKH (Suppl. Figure S1B, Information). By testing various related insect AKHs, Marco
Supporting Information, and Table 1). Compared with dec- et al. found that in fact the Leu2 to Val2 exchange already leads
apod RPCH, DappuAKH has three aa-exchanges in positions 2, to a substantial loss of RPCH-like activity, which is virtually lost
Figure 4. MALDI-TOF/TOF fragmentation spectrum (direct profiling) of Dappu allatostatin B3 (DappuASTB3, black letters on white background) at
m/z 1112.6 and DappuRYamide 1 (RYa1; white letters on black background) at m/z 1115.5 from a brain preparation. Prominent y- and b-fragments
from both ion signals are labeled. The fragments were analyzed manually to confirm the predicted amino acid sequence of DappuASTB3 as well as of
DappuRYamide 1 in the sample that could otherwise not be separated due to mass similarity.
when, in addition, exchanges such as Gly7 or Pro7 to Ser7 or The 5-exon Dappu astB-gene is much more complex than the
Thr7, respectively, occur as is the case in DappuAKH.24 corresponding 2-exon genes in D. melanogaster (CG6456) and T.
castaneum (GLEAN_07661). It gives rise to a single mRNA.
Allatostatin-A (ASTA) Type Peptides Beginning with an SP of 20aas, the open reading frame (ORF)
In crustaceans, up to 40 different allatostatin-A (ASTA) type spans exons 1 to 5 and encodes seven different typical ASTB type
peptides were identified from crab, crayfish and prawn CNSs,1012 peptides with a W(X)6Wamide motif, a single DappuASTB2
some of which even arose from post-translational processing and with an unusual W(X)7Wamide motif (Figures 1B,C and 3A),
modification.12 Even more ASTA peptides were found in several and three PRPs. We identified five W(X)6Wamide-type peptides
crustaceans with the aid of transcriptome mining and mass by fragmentation-sequencing (Table 1, Figures 3B and 4). To
spectrometric analyses.13 Whereas in insects, a maximum of 14 our knowledge, none of the DappuASTB peptides is identical to
different ASTA peptides can be derived from a single mRNA any known crustacean or insect ASTB-type peptides. However,
precursor,25 the three known crustacean ASTA-precursors yield ASTB 68 show very close similarities to insect ASTB isoforms
up to 29 different peptides, as revealed in crayfish, shrimp and a by sharing not only the typical W(X)6Wamide motif but up to a
copepod.2628 maximum of six identical and up to even seven closely related aas.
The 3-exon Dappu astA-gene is the first unraveled crustacean Only DappuASTB5 shows higher aa-identities with lobster
gene for ASTA-type peptides and fully supported by several HomamASTB than with insect ASTB, but only three identical
ESTs and our RT-PCR results (Suppl. Table S2, Supporting residues can be found when comparing the DappuASTB se-
Information). It encodes single copies of six different Dappu quences with that of the longest C. borealis CanboASTB31 as
ASTA peptides with a typical C-terminal F/YxFGLamide motif, detailed in Figure 3C.
and three precursor-related peptides (PRPs) separated by R or
KR-cleavage sites (Suppl. Table 1, Supporting Information). We Allatostatin-C (ASTC) Peptides
have identified four of the six DappuASTA peptides (Figures 1A, Allatostatin-C (ASTC) peptides were first discovered in
B,D and 2A) by fragment analysis (Table 1, Figure 2B). This Manduca sexta. They share the common sequence motif
small ASTA peptide diversity in D. pulex is much lower when (X)5C(X6)CF which can be C-terminally amidated or not. They
compared with the multitude of ASTA peptides on all known are derived as a single copy from precursors giving rise to mature
decapod and many insect ASTA-precursors.29 Interestingly, peptides usually carrying either a nonamidated (X)6CYFN-
however, all six DappuASTA peptides are different from those PISCF -often N-terminally blocked by pyro-Glu- or a SxWKQ-
of decapods and insects with regard to one or more aas among CAFNAVSCFamide as distinctions of this motif.32 Recently, it
the first 319 aas before the C-terminal pentapeptide motif. was established that pairs of paralogous astC- and closely related
so-called astCC-genes form head-to-tail clusters on the chromo-
Allatostatin-B (ASTB) Type Peptides somes of several insects from very different systematic groups.33
Allatostatin-B (ASTB) peptides are well-conserved nonapep- The derived AST-C/AST-CC type peptides normally belong to
tides sharing the characteristic sequence motif W(X)6Wamide either the mentioned subtypes or further N-terminally extended
and some weak similarity to vertebrate galanin. These peptides forms. In decapod crustaceans, the tetradecapeptide SYWKQCAF-
exhibit myoinhibitory, juvenile hormone, and ecdysteroid bio- NAVSCFamide and the pentadecapeptide CanboASTC1 pQIR-
synthesis-inhibiting effects in insects.30 VPNDWAHFRGSWa- YHQCYFNPISCF were recently found well conserved in a large
mide was the first neuromodulatory ASTB-type peptide variety of species3436 and recently also as EST-predictions from
identified in the crab C. borealis followed by a number of similar D. pulex.37 SYWKQFNAVSCFamide has both neuromodulatory
decapod crustacean peptides found by mass spectrometry.13 (in the crab stomatogastric system) and cardioactive properties.34
Figure 5. Four paired clusters of D. pulex neuropeptide and protein hormone genes without intervening genes in tandem arrangements and their
derived mRNAs. (A) Dappu astC2/astC1-gene cluster showing tandem head-to-tail arrangement of two similar genes with the positions of the respective
DappuASTC1/C2 (A) codons (note that the intergenic space has been truncated for clarity). (B) Dappu bursicon B and A (bursB/A; BB, BA)-genes in a
head-to-head arrangement only 370bp apart. (C) Tail-to-tail orientated tandem of long and short Dappu eclosion hormone (ehl/eh)-genes. (D) Two
glycoprotein hormone-encoding Dappu gpa2- and Dappu gpb5-genes in tail-to-tail arrangement. Note the only 6 bp-long stretch between both genes.
Light-gray shading, S or SP signal peptide; medium dark-gray shading, coding regions; dark-gray shading, identified peptide/protein hormone coding
regions; very light-gray shading, 50 and 30 untranslated regions; e, exon; i, intron (superscript numbers 02 indicate intron phase); bp, base pairs; asterisk
indicates stop codon; >,< plus, minus strand reading direction.
Figure 6. DappuASTC13 peptides and alignment with insect (green) and decapod (dark blue) crustacean ASTCC/C peptides. (A) Alignment of the
sequences of the novel DappuASTC2 and the DappuASTC1 peptides (as arranged in tandem in the gene cluster, see Figure 5A) shows strict
conservation of the cystine-bridges and the Cys-flanking aas with those in ASTCs/ASTCC tandems from insects. Note higher numbers of aa-identities in
DappuASTC2 and ASTCC-like peptides compared with those in the ASTC1/ASTC-peptides. (B) DappuASTC13 peptides aligned to each other
show all hallmark aas with lowest consensus aas among each other (bold-faced) in between and next to the cystine-bridges but distinct differences at the
N- and C-termini. (C) DappuASTC3 is more similar to the decapod crustacean CanboASTC135 and locust ASTCC than to the DappuASTC1 and
DappuASTC2 and has the largest consensus. (Insect ASTC/CC sequences from Veenstra, 200933).
Similar gene, precursor and peptide structures were found for identical to the peptide widely conserved among decapod crusta-
three astC13-genes in the D. pulex genome. Our current gene cean and some insect groups.34 DappuASTC2 (GQSSQRVFW-
models clearly comprise a gene cluster of the 3-exon Dappu RCYFNAVSCF) closely resembles several of the insect AST-CC-
astC2-gene followed downstream by the revised 4-exon Dappu like peptides, but such peptides are not known from other
astC1-gene spanning about 5.2kB (5227bp) on scaffold 9 crustaceans. The third Dappu astC3-gene has been high-score
(Figure 5A). This gene cluster is an intermediate between the annotated as a 3-exon gene on a different scaffold 26 (Suppl. Table
known Drosophila and Tribolium AST-CC/C gene clusters. The S1, Supporting Information). Its intron-2 splits the coding region
peptides derived from this gene cluster are either highly similar or of the peptide DappuASTC3 with the predicted sequence SKQL-
identical to several known ASTC peptides from insects33 and RYHHCYFNPISCFOH, which is much more similar to crab
crustaceans (Figures 5A and 6A). All DappuASTC13 peptides CanboASTC1 than DappuASTC1 and DappuASTC2 (Figure 6C).
have a similar core structure with a consensus x--C-FN-xSCF We have so far only been able to identify the fragment ASTC3-PRP-
(Figure 6B). DappuASTC1 (SYWKQCAFNAVSCFamide) is 11142 by mass spectrometry (Suppl. Table S3, Supporting
middle part of the peptides to an AT-like L. migratoria abdominal

ganglia myotropin-1 LocmiAGMT141 and moth M. sexta AT,
which are larger in the cases of chelicerate (tick Ixodes scapularis)
or mosquito AT (Figure 7C). This is the first AT identified in
crustaceans; and it came to our surprise, that decapod crusta-
ceans do not seem to have an AT-like peptide since we did not
find any even by sophisticated BLAST searches.
Bursicon
The protein hormone bursicon is known for more than 80
years as the cuticle tanning and sclerotization factor of insects. It
occurs in insects as a heterodimer or homodimer of bursicon-A
and/or bursicon-B subunits. Both subunits are large N-glycosy-
lated proteins with typically eleven cysteines, ten of which are
involved in the formation of cystine-knots.42 Bursicons occur
ubiquitously in all hitherto analyzed insects, similarly in some
decapod crustaceans and Daphnia arenata, and perhaps even in
some chordates, but are absent from nematodes.4345
The D. pulex genome contains separate bursA- and bursB-
genes, one for each bursicon subunit, that lie only 370bp apart to
form a head-to-head inverted tandem (Figure 5B). The Dappu
bursA-gene shows the best fit with currently available ESTs
especially with regard to the SP and the mature peptide ORF
(Suppl. Figure S2A, Supporting Information). Although several
insects do also have 3-exon-genes, this cluster formation of the
Dappu bursA and bursB subunit genes appears unique. According
to Robertson et al. 2007,46 the water flea bursA-gene may have
lost the first phase-1 intron (now a phase-0 intron) that the
Figure 7. Novel Dappu allatotropin (AT) gene, the derived DappuAT authors considered characteristic for all insects. However, the
and comparison with insect and chelicerate allatotropins. (A) The Dappu bursB-gene has retained such phase-1 intron (Figure 5B).
2-exon gene (upper panel) encodes DappuAT (A) flanked by two While both bursicon subunit mRNAs of the two species D. pulex
precursor-related peptides (PRP), the second being split by the intron and D. arenata44,45 are almost identical (one silent base pair-
(shadings as in Figure 5). (B) Fragmentation mass spectrum (direct exchange in the bursA-ORFs, three more outside), their pre-
profiling) of DappuAT at m/z 1323.7 with labeled y- and b-fragments
cursor-derived protein hormones are completely identical.
and immonium ions. (C) DappuAT shows high similarities to allato-
tropin-like peptides from hemimetabolous (L. migratoria accessory Alignments of DappuBursA and DappuBursB with decapod
gland myotropin LocmiAGMT140) and holometabolous insects crustacean and insect sequences show a high degree of simila-
(green; M. sexta ManseAT, A. gambia AnogaAT154), but also to two rities especially with regard to the cystine-knot part (Suppl.
allatotropin-isoforms from the chelicerate Ixodes scapularis (EEC06620; Figure S2A,B, Supporting Information). Most likely a gene
orange). duplication event gave rise to this gene cluster.
Information), which, however, confirms the expression of the CCHamide

ASTC3-precursor in the D. pulex brain. Originally described as a parturition hormone with the
sequence GCLSYGHSCWGAHamide in the viviparous Tsetse
Allatotropin fly Glossina palpalis,47 a related peptide, GCAMFGHSCYGA-
Allatotropins were originally discovered in a moth as stimu- Hamide was later discovered in the genome of the silk moth
lators of juvenile hormone production in corpora allata but are B. mori and named CCHamide because of its cyclic nature brought
now considered multifunctional insect peptides with myotropic, about by a cystine-bridge and the C-terminal His-amide.48 We
cardio-excitatory and circadian clock-modulating activities.38 In have recently found that all insects with a sequenced genome
the CNS of different moth species, complex alternative splicing have two distinct CCHamides, CCHa1 and CCHa2, derived
processes can lead to up to three different mRNAs derived from a from two different genes, and that each activates its own specific
single gene, which tissue-specifically encode up to three different receptor.49
peptide splice forms.39,40 In the D. pulex genome we have only found one CCHamide
In D. pulex, one single 2-exon Dappu allatotropin (at)-gene gene and not two as in insects. This gene gives rise to a 106aas
yields a novel AT-like peptide DappuAT, the amidated trideca- long precursor from which DappuCCHamide (Table 1) is
peptide GFKTVGLATARGFamide, flanked by dibasic cleavage liberated directly after the SP via a signal peptidase and from a
sites (RR, KR) and two PRPs that are different in length (Suppl. C-terminal dibasic proprotein convertase cleavage site preceded
Tables S1, Supporting Information, and Figure 7A). We have by a Gly that allows for an amidation (Suppl. Table S1,
confirmed the exon/intron structure of the precursor by RT- Supporting Information). This precursor structure is a hallmark
PCR (Suppl. Table S2, Supporting Information) but we did not of CCHa1-type peptides. The CCHa2-type peptides all derive
find any products of alternative splicing. We sequenced the from precursors with dibasic cleavage sites at both ends of the
peptide by fragmentation mass analysis (Table 1; Figures 1C peptides.49 When aligned with some selected insect CCHamide-
and 7B). DappuAT shows only minor sequence differences in the 1 and -2 subtypes, DappuCCHamide is intermediate between the
two forms with slightly higher similarities to CCHa1-type Diuretic Hormones DH31 and DH52
peptides (Suppl. Figure S3A,B, Supporting Information). Apart from CAPA/PVK-related peptides (see also below) and
insect kinins (not found in the D. pulex genome), two other
Corazonin families of C-terminally amidated peptides are known as diuretic
The N- and C-terminally blocked undecapeptide corazonin, hormones (DHs), namely calcitonin-like DHs (CLDHs) with
pQTFQYSRGWTNamide (Arg7-CRZ), is known from many 3037aas and corticotropin-releasing factor (CRF)-like DHs
insect species.50 In locusts, however, a His7-CRZ isoform controls with 4047aas in a large variety of insects.68 In crustaceans, so far
behavioral and morphological transitions during swarm formation only one 31aas-long member of CLDHs has been identified for
and migration.5153 However, Arg7-CRZ is more ubiquitous with the lobster Homarus americanus.69 CLDHs have a well-conserved
pleiotropic functions related to the initiation of ecdysis behavior54 structure of 31aas, therefore often being called diuretic hormones
and stress control55,56 in insects, but CRZ-genes are lacking in some 31 (DH31). Furthermore, we provide here the first evidence for
insects such as beetles and aphids.57,58 Arg7-CRZ is so far the only the existence of a CRF-like DH in crustaceans.
isoform detected in decapod crustaceans,59,60 and single ESTs are The Dappu dh31-gene is a 3-exon gene in which all exons
known for Arg7-CRZ-precursors of shrimp L. vannamei60 and water contribute to the DappuDH31-precursor. These structures
flea Daphnia carinata.61 clearly resemble those of the Drome dh31-gene (CG13094),
The Dappu crz-gene is a 3-exon gene which encodes a 133aas which, however, contains two more exons 50 upstream separated
long precursor containing the peptide DappuArg7-CRZ directly by a large intron (10330bp) not found in D. pulex. We have
following the SP (Suppl. Table S1, Supporting Information). confirmed the exon/intron-structure of the ORF by RT-PCR
We fully identified this peptide as N-terminally blocked (Suppl. Table S2, Supporting Information) and the sequences of
by a pyro-Glu using fragmentation sequencing (Table 1, DappuDH31 together with the PRP-1 SLLRNSDYEHQ by
Figure 1B). Apart from DappuCRZ, however, the insect, fragment analysis (Table 1, Suppl. Table S3, Supporting Infor-
decapod and DappuCRZ precursors have only little in com- mation, Figure 1A,C). The sequences of DH31s from isopod and
mon except for the presence of cysteine residues in CRZ- decapod crustaceans and insects are extremely conserved as
precursor associated peptides (Suppl. Table S1, Supporting detailed in Suppl. Figure S4A (Supporting Information).
Information). In fact, these peptides are much less conserved The 4-exon Dappu dh52-gene, found on a different scaffold,
than AKH-precursor related peptides (APRPs) which may gives rise to a 191aas long precursor with a 25aas long SP that
indicate a more rapid evolution of the crz-genes. liberates the novel 52aas long amidated DappuDH52 and three
Crustacean Cardioactive Peptide smaller PRPs (Suppl. Table S1, Supporting Information). Dap-
In all hitherto investigated arthropod groups, crustacean puDH52 is the first crustacean CRF-like DH and shows closest
cardioactive peptide (CCAP) is an extremely conserved cyclic similarities in length and overall structure to beetle TricaDH47,
nonapeptide PFCNAFTGCamide with an intramolecular disul- but is also quite similar to DH46s from hemimetabolous and to
fide bridge.6 CCAP is a well-known key player in ecdysis DH44s from holometabolous insects (Suppl. Figure S4B, Sup-
regulation in insects and crustaceans.6264 porting Information).
As unraveled here for the first time in crustaceans, there is a Ecdysis-Triggering Hormones
single 3-exon Dappu ccap-gene that encodes the nonapeptide A single 4-exon Dappu eth-gene in the D. pulex genome
DappuCCAP typically flanked by a dibasic and a tribasic proces- encodes a precursor with two ecdysis-triggering hormones
sing site. This novel isoform of CCAP, PFCNAFAGCamide, (ETHs) in tandem, DappuETH1 and DappuETH2, and two
with Ala7 exchanged for Thr7 (Table 1; Suppl. Table S1, long PRPs (Suppl. Table S1, Supporting Information). The
Supporting Information) has a primary structure that is unusual structures of gene, precursor and peptides closely resemble insect
among crustaceans and insects, since in all hitherto investigated ETH genes and gene products. Alignment with other insect
arthropod groups, no CCAP isoform other than the original one ETHs shows that both ETH-peptides bear the typical ETH-
has ever been found. Much more different isoforms are only signatures, that is, PRXamide (X= I, L, V, M) C-termini.48,70
known from some worms and mollusks.6,65 The DappuCCAP- DappuETH2 is, however, much longer than insect ETHs (Suppl.
precursor structure, however, resembles those of decapods and Figure S5, Supporting Information). To date there are no
insects.66 Furthermore, the overall Dappu ccap-gene structure decapod ETH-peptides known.
itself is remarkably similar to ccap-gene structures of M. sexta,67
D. melanogaster (CG4910) and T. castaneum (LOC664330), Eclosion Hormones
except that the Manse ccap-gene has 50 upstream a fourth The D. pulex genome contains two genes, the Dappu eh- and
additional exon followed by an intron interrupting the SP. Dappu ehl-genes, each encoding a single eclosion hormone (EH)-like
peptide. Both genes occur as a tail-to-tail inverted tandem cluster
DENamides right next to each other on the same scaffold. DappuEHL is with
The novel Dappu dena-gene gives rise to a precursor that liberates unusual 113aas about twice as long as DappuEH (56aas, Figure 5C,
at least nine closely related previously unknown peptides which we Suppl. Table S1, Supporting Information). By use of RT-PCR we
named DENamides: five copies of the decapeptide FMGLSHFDE- confirmed the predicted structure of the DappuEHL precursor
Namides, two copies of Lys2-DENamide, FKGLSHFGENamide, especially with regard to the exon/intron positions (Suppl. Table S2,
and another related peptide KCHFDENamide and the C-terminal Supporting Information). DappuEH strikingly resembles insect EH
FMGLSHFHTKTWTOH (Suppl. Table S1, Supporting In- peptides. It bears with several insects up to 53% of sequence identity
formation). It was found by BLAST searches using invertebrate (e.g., with beetle TricaEH; 71% sequence similarity) while a
FMRFamide-related peptide precursors (e.g., from Mytilus edulis decapod EH from the crab C. sapidus and from the chelicerate
CAA10949) with which it mainly shares similarly staggered KRFM I. scapularis exhibit less similarity as detailed in Figure 8. The
sequences. Otherwise, DENamides have no resemblance with any DappuEHL structurally resembles DappuEH and other arthropod
other known arthropod peptide. EHs with regard to signature parts such as the positions of the first
Figure 8. Dappu eclosion hormones (DappuEH and EHL, underlined) aligned with other arthropod EHs. Except for the long EHL isoforms of the
branchiopod crustaceans D. pulex and horseshoe shrimp Triops cancriformis (FM869498; dark blue), all EHs show close structural similarities extending
not only to the positions of cysteines (shaded red) but also to a similar helix-motif (h) character as worked out recently for ManseEH.155 DappuEH
pertains, however, lowest similarity to the EH from the crab Callinectes sapidus (CalsaEH CV224237; dark blue). The overall aa-similarities of
DappuEHL with DappuEH, with insect EHs (below, green), and the decapod EH (above) are low, slightly higher only with long TriopsEHL.
DappuEHL has a signature similar to that of DappuEH but has an additional 16aa long stretch between the last two of the six cysteines and a much longer
C-terminus; the latter feature it shares with the Triops EHL. (Insect EHs from: beetle Tribolium castaneum XP_969164, pea aphid Acyrthosiphon pisum
XM_001943582, mosquitoes Aedes aegyptii CH477404.1, Anopheles gambiae XM_001230804, fruit fly D. melanogaster CAA51051, moths Bombyx mori
NM_001043842, Manduca sexta M27808.1, Asian corn borer Ostrinia furnacalis ABG66962.1; chelicerate EH from: Ixodes scapularis EH
XM_002399230, orange).
five cysteines and sequence stretches in between. However, Dap- and known only from a couple of insects such as the beetle
puEHL has only 29aas identical to DappuEH and bears a glycine- T. castaneum (TricaFaRP, GLEAN_07769) and the cockroach
rich 16aas longer stretch between the last two cysteines and a 40aas Periplaneta americana71 but more common in nematodes such
longer C-terminus than DappuEH (Figure 8). The overall simila- as C. elegans (three flp-genes with 12 FIRFamides72). Of note
rities of DappuEHL with DappuEH, other insect EH, and a single for comparison, in insects, the PeramFaRP-precursor encodes
decapod EH are low but slightly higher only with a novel EHL from 10 different FIRFamides but no FLRFamide,71 whereas the
the branchiopod Triops cancriformis, with which DappuEHL also TricaFaRP precursor encodes a mixture of FIRFamides and
shares a similarly elongated C-terminus (Figure 8). This FLRFamides. Other FIRFamides in B. mori are the prothor-
Dappu ehl-/Dappu eh-gene cluster may have resulted from a acicostatins (BommoFMRFamides BRFa-1 to -4) that regu-
gene duplication. late ecdysteroidogenesis in silkworm prothoracic glands.73
Extended FMRFamides Glycoprotein Hormone GPA2/GPB5
The Dappu firfamide-gene is the first extended FMRFamide Further members of the cystine knot-containing glycopro-
gene unraveled in crustaceans, encoding two DappuFIRFa- tein hormone family are essential for gonadal and thyroid
mides and six PRPs (Suppl. Table S1, Supporting In- functions in all vertebrates, and may have many roles in
formation). We identified the mature FIRFamides, including development. The glycoprotein hormone alpha- (GPA-) sub-
an N-terminally truncated variant, and two PRPs by mass unit forms heterodimers with different beta- (GPB-) subunits in
spectrometry (Table 1, Suppl. Table S3, Supporting Informa- mammalian gonadotropins (LH and FSH) to activate respec-
tion, Figures 1A,C and 9A). The peptides are flanked by tive receptors. Two novel subunits recently discovered in
mono- or dibasic Arg-cleavage sites (R; RR) and an excep- humans form a heterodimer called thyrostimulin, consisting
tional monobasic Lys-cleavage site occurring between the fully of alpha- (GPA2) and beta-subunits (GPB5), activate the
sequenced amidated 27-mer PRP-3 and DappuFIRFa-2 thyroid-stimulating hormone (TSH)-receptor. 74 Similar
(Table 1, Suppl. Tables S1, S3, Supporting Information). molecules have been discovered in lower chordates and inverte-
FIRFamide-encoding genes are relatively rare in arthropods brates including insects and nematodes.48,75
similarities were found for the body louse Pediculus humanus,

dipteran and beetle glycoproteins (Figure 10A,B). BLAST searches
did not find any identified or predictable GPA2/GPB5 in
decapod crustaceans.
Inotocin
Oxytocin (OT)- and vasopressin (VP)-like peptides are phylo-
genetically old and highly conserved multifunctional nonapeptides
widespread among proto- and deuterostomian lineages.76,77 In
insects a VP-like peptide was originally isolated from L. migratoria78
and twenty years later VP-like preprohomone and receptor genes
were identified in the beetle T. castaneum.77,79 It is interesting,
however, that genes for insect oxytocin/vasopressin-like peptide
(inotocin) and its receptor appear to have been lost in a number of
holometabolous insects.77,79 Bioinformatics analyses of the D. pulex
genome showed that also crustaceans contain an inotocin-like
peptide, CFITNCPPGamide, with the typical cystine-bridge but
with the unprecedented occurrence of a proline in position 8.77
Here, we provide a revised version of the Dappu-inotocin (it)
gene as confirmed by RT-PCR (Suppl. Table S2, Supporting
Information). Its gene structure is typical for all hitherto known
OT/VP-like peptides encoding genes76 including those recently
discovered in holometabolous insects57,77,80 (Figure 11A). The
gene gives rise to a precursor similar to those from other OT/VP-
like peptides: the SP is directly followed by the encoded
DappuIT, and a C-terminal neurophysin domain. The latter
contains 14 cysteine residues aligning well with neurophysins
from other OT/VP-like precursors likely giving rise to seven
cystine-bridges (Figure 11B).
Insulin/Insulin-like Growth Factor (IGF)-Related Peptides
Invertebrate insulin-related peptides (IRPs) are derived from
multigene families that are expressed in the brain and peripheral
tissues. These peptides can serve as hormones, growth factors or
neuromodulators.81 Insulin-like peptides in arthropods were first
discovered in B. mori as bombyxin,82 which turned out to be just
one out of many similar IRPs encoded by 38 bombyxin genes in
B. mori; this extreme is not found in other insects, which have
only one IRP-gene as in locust species or up to 78 IRP-genes as
in dipterans.81 In crustaceans, insulin-related peptides are so far
known only as androgenic gland hormones (AGHs) responsible
for differentiation and determination of sex. First AGHs were
Figure 9. MALDI-TOF/TOF fragmentation spectra of FMRFamide
identified in woodlice as complex N-glycosylated clearly insulin-
related peptides of D. pulex. (A) DappuFIRFamide 1 at m/z 1208.68,
(B) Dappu myosuppressin (MS) at m/z 1257.64, and (C) Dappu related androgenic gland peptides (IAGs) that elicit male
sulfakinin 2 (SK2) at m/z 1301.54 from direct profiling of brain phenotypes83,84 and later also in decapod crustaceans.85,86 Hall-
preparations. Prominent y- and b-fragments and immonium ions are marks of these IRPs include the typical sequence of B-, C-,
labeled. The fragments were manually analyzed and confirmed the A-chain peptides with distinct cleavage sites (KR, RxxR) and
predicted amino acid sequences. putative N-linked glycosylation motifs (NxS/T). In all IRPs
except for IGFs, the C-peptide is cleaved from the precursor
after the canonical cystine-bridges have formed.
For crustaceans, we provide here the first evidence for Here, we describe four different single genes encoding one typical
corresponding genes and separate derived protein hormone IRP, and three insulin-like growth factor/relaxin-related peptides in
subunits in the D. pulex genome. The Dappu gpa2-gene and the the D. pulex genome. Originally, DappuIRPs14 were numbered in
Dappu gpb5-gene are both 3-exon genes clustered tail-to-tail series of their first annotation by us. The 5-exon Dappu irp2-gene
right next to each other (only 6bp apart) on the same scaffold gives rise to the DappuIRP2-precursor that resembles most closely
(Suppl. Table S1, Supporting Information; Figure 5D). They the insect insulin precursors, especially those of locust IRP and some
give rise to large precursor-derived protein hormones of bombyxin precursors (Figure 12A,B). This gene structure appears
118aas (DappuGPA2) and 133aas (DappuGPB5) that have unique since insulin genes are normally 3-exon genes, only IGF-
10 cysteines involved in the typical cystine-knot formation (for genes have 45 exons.87,88 The DappuIRP2-ORF spans four exons
details see: Figure 10A,B). Precursor comparisons show that and the coding regions of the B-chain and the C-chain are
DappuGPA2 and DappuGPB5 are closely related to well-conserved interrupted by introns 3 and 4, respectively, as confirmed by RT-
glycoprotein heterodimers in insects, and to some extent to PCR (Suppl. Tables S1, S2, Supporting Information). By mass
mouse and human glycoprotein hormones. Interestingly, highest spectrometry, we identified a B-chain C-terminally flanked by a
Figure 10. Alignments of (A) DappuGPA2- and (B) DappuGPB5-subunit precursors with those of other arthropod and mammalian cystine-knot
proteins. Similarities are higher in the cases of GPA2 than of GPB5 proteins. Most highly conserved are the actual cystine-knot sequences embraced by
the first and the tenth cysteine with some parts of lower conservation between the fourth and the fifth cysteine in the GPA2s or the fifth and the sixth
cysteine in the GPB5s (asterisks indicate ring parts with conserved aas of the cystine-knot built by cysteines 37 and 48). Insect (green) GPA2, GPB5
from: mosquitoes Anopheles gambiae AnogaGPA2 XM_317164.4, AnogaGPB5 XM_555160.3, Culex quinquefasciatus CulquGPA2 XM_001863270.1,
CulquGPB5 XM_001863269, moth B. mori BommoGPA2 NM_001130903.1, BommoGPB5II BN001262.2, fruit flies Drosophila virilis DroviGPA2
XM_002051238.1, DroviGPB5 XM_002051240.1, DromeGPA2 AY940435.1, DromeGPB5A NM_001110865.2, louse Pediculus humanus PedhuG-
PA2 XM_002427773.1, PedhuGPB5 BN001256.1; beetle TricaGPA2 NM_001170773.1; TricaGPB5 BN001258.1; mammalian (gray) GPA2, GPB5:
Homo sapiens HomsaGPA2 BC093962.1, HomsaGPB5 AF467770.1, mouse Mus musculus MusmuGPA2 NM_130453.3, MusmuGPB5 NM_175644.3;
∼ indicate ends of SPs.).
rare monobasic Lys-cleavage site but also two short PRPs directly does not have any N-glycosylation sites, and when aligned with
upstream from the B-chain on the precursor (Table 1, Suppl. decapod and isopod crustacean IRPs distinct differences are
Tables S1, S3, Supporting Information, Figure 1A,D). Interest- detectable. These differences comprise not only the lack of an
ingly, the DappuIRP2-PRP-2 exhibits some similarity to a so-called additional cystine-bridge as in isopod AGHs but also an additional
locust IRP-precursor-related copeptide identified previously as the amino acid between the cysteines of the B-chain and a 4aas-gap
glycogenolysis-inhibiting peptide89 (Figure 12C). DappuIRP2 between the second and the third cysteine in the A-chain.
Figure 11. Comparison of oxytocin/vasopressin-like genes and preprohormones of D. pulex, other invertebrates and mouse. (A) The 3-exon genes for
DappuInotocin (DappuIT), jewel wasp Nasonia vitripennis inotocin (NasviIT; NW_001819015), and mouse Mus musculus oxytocin (OT;
OTTMUSG00000015526) are highly similar; note that both introns interrupt the neurophysin (NP) sequences twice in similar positions in all genes. (B)
DappuIT and other oxytocin/vasopressin-like preprohormones from insects (green), mollusks, an earthworm (Eisenia fetida annetocin EisfoAT; BAA36458)
and a vertebrate (Mus musculus; gray) align particularly well with regard to the oxytocin-like nonapeptides and the 14 cysteines of the neurophysins. Note longer
stretches in the DappuIT-precursors flanking the first and the second neurophysin domains containing four and three cystine-bridges, respectively. (Insect ITs
from: T. castaneum TricaIT GLEAN_06626, NasviIT NW_001819015; snails: Aplysia kurodai Lys-conopressin AplkuLCP BAB40371.1, Lymnaea stagnalis
Lys-conopressin LymstCP AAA29289.1); intron positions are indicated by triangles, ∼ and diamond indicate ends of SPs.
However, the DappuIRP2 precursor clearly shares the latter two derived precursors are most similar to each other (49% amino
characters with typical insect IRPs and a molluscan IRP (Suppl. acid identity, 62% conservation score) but also to the Dap-
Figure S6, Supporting Information). puIRP1 precursor (Figure 12D). When applying the rules for
The 4-exon Dappu irp1-gene encodes a precursor that does insect peptide hormone precursor cleavages,19 the DappuIRP3-
not have a C-peptide but a single putative dibasic cleavage site precursor resembles typical IGF-like precursors, in which the
(RR) between the very long B-chain (50aas) and possibly a 24aas short C-peptide is not cleaved, since it has only unlikely dibasic
A-chain, if the Arg110 downstream of the last cysteine is used as a (RK) and monobasic (R) cleavage sites.
cleavage site. Thus, the precursor and the peptide are more
reminiscent of D. melanogaster DromeILP6 and similar ILPs of Ion Transport Peptides
other dipteran and hymenopteran insects than related to any Previous studies showed that water fleas Daphnia magna do
decapod AGH except for the occurrence of a typical N-glycosyla- not have a typical XOSG-system but only brain CHH-interneur-
tion site (NVS) at the beginning of the B-chain (Figure 12D, ons and peripheral CHH-ir neurons.91 The latter neurons are
Suppl. Table S1, Suppl. Figure S6, Supporting Information). If reminiscent of shore crab pericardial organ (PO) neurons
not cleaved at Arg,110 there would be even two more such expressing one of two alternative splice forms derived from a
N-glycosylation sites (NQT, NES). Because of the lacking 4-exon CHH-gene encoding a 72 aas long amidated “short” sinus
C-peptide, the long C-terminus with putative D- and E-domains gland XOSG-CHH and a nonamidated 73aas POCHH.92
and the similarity to DromeILP6, DappuIRP1 should thus be Similar alternative splice forms also occur for several insect short
considered IGF-like88,90 as is likely also the case for the remaining and long ion transport peptides (ITP/ITPLs), originally discov-
DappuIRPs. The Dappu irp3- and irp4-genes are obvious para- ered as antidiuretic factors in locusts,93 and are expressed by
logs of the Dappu irp1-gene and both have even more similar the Dappu itp-gene. We used these names here instead of
4-exon gene structures and occur about 102kb apart from each CHH/CHHL to adequately refer to their striking sequence
other in tail-to-tail arrangement on the same scaffold 18. The similarities with ITP/ITPLs from insects (Suppl. Figure S7,
Figure 12. Dappu insulin-related peptide-2 (irp2) gene and comparisons of its gene products with insect IRPs and other DappuIRPs. (A) Complex 5-exon
structure of the Dappu irp2-gene shows a long stretch between the SP and two identified precursor-related peptides (PRPs: 1, 2) preceding the
B- and C-chains. The latter are both split by introns i3 and i4, respectively. The B- and A-chains of DappuIRP2 are closely related to those of insect IRPs
(green). (C) DappuIRP2-PRP2 shows some similarities to locust glycogenolysis-inhibiting peptide (GIP89). (D) DappuIRP1, -3, and -4 precursors are very
similar, particularly in the cases of DappuIRP3 and DappuIRP4 (note the DappuIRP3:4 comparison; putative cleavage sites in blue). Triple asterisks indicate
possible N-glycosylation sites at NxS sites in DappuIRP1, -3, -4 or a NxT site in DappuIRP1; diamond indicates end of SP.
Supporting Information). Two further genes in the D. pulex ITP-sequences, and the first half (here: aas 140) of the ITP in
genome encode clearly ITP-related but more elongated the mRNA-parts are derived from the second exon as is the case
peptides. for all short and long CHHs and ITPs; the alternatively spliced
The Dappu itp-gene is a type-I CHH/ITP 4-exon gene.2,93 It exons 3 plus 4 or exon 4 encode the second half of all CHHs/
gives rise to two mRNAs encoding either a short or a long ITPs, respectively.2,93 The same is the case for the 4-exon Dappu
alternative splice form of DappuITP (72aas) or DappuITPL itp-gene, in which exon-2 encodes a 12 aas DappuITP-PRP
(79aas), respectively, that we have confirmed by RT-PCR (MSALSSGHHSLS) that is similar in length to many insect
(Suppl. Table S2, Supporting Information). Only the short ITP-PRPs but much shorter than in most decapod CHH-
DappuITP has an amidated C-terminus. Both peptides lack the precursors3,93 (Suppl. Figure S7AC, Supporting Information).
N-terminal pyroglutamate protective group characteristic for Two further CHH/ITP-like genes, the Dappu itpln-gene and
most decapod crustacean CHHs,2,14 but instead begin with a the Dappu itpn-gene, exhibit clear-cut type-I CHH/ITP gene
SFF-group as is typical for many insect ITPs. Further character- characteristics2,93 (Suppl. Figure S7A, Supporting Information),
istics typical for all CHH-like ITP-precursors are the occurrence although these D. pulex genes are unusual 4-exon and 2-exon
of a short SP, a short part of it usually being encoded by the first genes, respectively. Derived precursors of both genes do not
exon.2,93 The rest of the SP and a ITP-PRP, which precedes the contain an ITP-PRP but instead exhibit elongated N-terminal
sequences following right after the predicted SP (Suppl. Figure conserved feature apart from the mature peptide part itself.
S7B,C, Supporting Information). The DappuITPLN-precursor DappuMS (pQDVDHVFLRFamide) is identical to the myo-
is, thus, a 99aas-long isoform that contains typical CHH-sub- suppressin from cockroaches, locusts, honeybee, and beetle, but
family motifs2 but a very different N-terminus with a KH- differs by one aa from the MS of decapods (Leu3, H. americanus,
sequence in a position in which one would have expected a KR GQ303179, P. clarkii, BAG68789.1) and dipteran insects (Thr1,
processing site (Suppl. Figure S7B, Supporting Information). A. gambiae, AGAP001474-PA, D. melanogaster, CG6440-PA).
Similar exchanges may be represented by the QQ-site instead of a
Neuroparsin
KR in an EST previously claimed to encode a MIH (EST
Neuroparsins (NPs) belong to a family of structurally related
FE418183) which instead clearly resembles ITPs (Suppl. Figure
large peptides (>100aas) of arthropods. NPs are characterized
S7C, Supporting Information). DappuITPLN hardly aligns with
by conserved positions of 1214 cysteine residues forming 6
products of type II-MIH/MOIH-like genes but much better with
cystine-bridges and share structural and functional similarities
long isoforms of DappuITPL and SchgrITPL (Suppl. Figure S8,
with vertebrate insulin-like growth factor binding proteins
Supporting Information; max. 35% vs 5557% identities, re-
(IGFBP).101103 NPs were discovered in Locusta migratoria
spectively). Another distinct feature of DappuITPLN is the lack
as products of pars intercerebralis neurosecretory cells of the
of a short DappuITP-like half of such splice-form usually
brain (hence the name) and are now known multifunctional
encoded by exon 4 in type-I CHH/ITP-genes.2 Thus, a clear-
neurohormones.101 While usually only one NP-precursor has
cut type II MIH/VIH/GIH-like peptide apparently does not
been found in many insects with annotated genomes,48,57,80
exist in the D. pulex genome contrary to a previous claim of the
except for sophophoran Drosophila species which lack NPs,103
possible existence of a DappuMIH.37 The ESTs encode a variant
locusts have up to four NP splice forms,101 but the Chagas-bug
with clearly DappuITP-like characteristics which, however, with
Rhodnius prolixus has even three np-genes.103 In crustaceans,
the above-mentioned N-terminal characteristics is not present in
NP-like peptides have so far only been predicted from ESTs for
the genome of D. pulex (Suppl. Figures S7C, S8, Supporting
some decapods and a copepod.5961,103
Information). Thus, D. pulex does neither have a MIH/MOIH-
In the D. pulex genome, there is one single 3-exon Dappu np-
nor a VIH/GIH-peptide with all the required molecular signa-
gene encoding a 78aas long neuroparsin with 14 cysteines
tures, that is, (i) a typical additional Gly at position 5 after the first
(allowing for seven putative cystine-bridges), as we have con-
cysteine and (ii) a Val in position 4 before the second cysteine
firmed by RT-PCR (Suppl. Table S2, Supporting Information).
when aligned with CHHs/ITPs, (iii) the N-terminal so-called
Strongest aa-identities with decapod and insect NPs occur in
A10 - and A50 -helix motifs,2 and (iv) typically 3-exon genes2
some core regions from the fourth to the thirteenth cysteine of
(Suppl. Figure S8, Supporting Information).
DappuNP. Overall aa-similarities reach 4954% but appear to be
The DappuITPN-precursor derived from the third CHH/
slightly higher when compared with decapod NPs (Suppl. Figure
ITP-like gene type clearly shows a putative deletion of the
S9, Supporting Information).
N-terminal 6aas SFFDIN (Suppl. Figure S7C, Supporting
Information). This precursor, on the other hand, perfectly Neuropeptide F
aligns with highest sequence identity with the DappuITP- Neuropeptide F (NPF)-related peptides are commonly con-
precursor and shares three distinct characteristics: (i) a SP sidered vertebrate neuropeptide Y (NPY) homologues (strictly
that is more than 2/3 identical, (ii) a nearly identical Dap- 36aas in vertebrates) containing precursor signatures such as the
puITP-PRP-like part (2 aa-exchanges only) and (iii) highest carboxy-terminal peptide of NPY (CPON), a PRP following NPY
identities from the first cysteine onward (6 aa-exchanges only; upon its precursor.104106 Almost all NPFs bear a C-terminal
Suppl. Figure S7C, Supporting Information). RPRFamide motif and two tyrosine residues at positions 9/10
and 17 from their C-termini.105,107 Recently, a novel definition of
Myosuppressin two different NPF-types (NPF1 and NPF2) has been proposed,
Myosuppressins are conserved arthropod decapeptides with which subdivides all known arthropod NPFs, some of which
the consensus motif X1DVX2HX3FLRFamide. These peptides may even arise from two different genes in a given species.106
have their own specific receptors that are not activated by The NPF2 peptides usually carry C-terminal GX1X2RYamide
above-mentioned FMRFamides.94,95 The first peptide of this motifs.107
type was discovered as a factor inhibiting hindgut-contraction The Dappu npf-gene gives rise to two mRNAs by alternative
in the cockroach Leucophaea maderae, named leucomyosup- splicing (Suppl. Figure S10A, Supporting Information) as evi-
pressin, pQDVDHVFLRFamide.96 Later on several isoforms denced by the occurrence of two distinct amplicons after
with Thr1-, Pro1-, or other substitutions were discovered in a RT-PCR (Suppl. Table S2, Supporting Information). The Dap-
variety of insects.97,98 To date, the only isoform identified in puNPF1 mRNA encodes a typical vertebrate NPY-like amidated
several crustaceans and known to decrease heart beat frequency DappuNPF1 of 39aas length followed by a DappuCPON of
in the lobster H. americanus is pQDLDHVFLRFamide.99,100 19aas in length. We have identified both peptides by mass
The Dappu ms-gene shows typically two exons and gives rise spectrometry (Table 1, Suppl. Table S3, Supporting Informa-
to an 112aas precursor (with 36aas SP) similar to those of tion, Figure 1A). A predicted D. magna NPF (EG565358108)
insects and decapods (Suppl. Tables S1, S2, Supporting In- is almost identical to DappuNPF1 but the former lacks the Gly3
formation). Fragmentation analysis confirmed the sequence of (Suppl. Figure S10B, Supporting Information). The Dap-
DappuMS (Figure 9B). Both the post-translationally modified puNPF1 splice form appears to be favored when looking at
pGlu-MS and the noncyclized Gln1-MS were identified by mass confirming ESTs37 and our RT-PCR data. The latter shows the
spectrometry and occur in a ratio of about 30/70% (Table 1, strongest amplicon corresponding to this mRNA (Suppl. Table
Figure 9B). In large parts of the insect and crustacean pre- S2, Supporting Information). Except for the existence of the
cursors aa-identities occur, especially two cysteines in the additional exon 2 in the Dappu npf-gene, its exon-intron struc-
precursor-related peptide directly after the SP stand out as ture is remarkably similar to that of the human npy-gene104
(Suppl. Figure S10A, Supporting Information). These close those of insects such as the honeybee A. mellifera, the beetle
similarities provide further support for the close relationship T. castaneum, the moth B. mori, and the mosquito A. gambiae.
between invertebrate NPFs and vertebrate NPY supposed However, the full length SP of the DappuOKII precursor is not
earlier.105 clear yet because of 50 upstream gaps in the genomic sequence.
As a second product of the Dappu npf-gene retaining exon 2, The exon/intron arrangement of the Dappu okII-gene, however,
the DappuNPF2 mRNA, encodes a 66aas-long DappuNPF2 as inferred by bioinformatics, is clearly EST-supported, at least
and the same 19aas-long DappuCPON as partly revealed by for the second exon (Suppl. Tables S1, S2, Supporting In-
RT-PCR (Suppl. Figure 10A, Suppl. Tables S2, S3, Supporting formation). Evidence from peptide alignments suggests that
Information). Shorter peptides such as a 37aas-long Dap- the DappuOKs are more closely related to the OK-tetradecapep-
puNPF2 fragment could be derived from the former protein, tides of insects, a copepod crustacean, and a chelicerate than to all
since it contains several monobasic cleavage sites. We were not OK-tridecapeptides of decapods. This is similarly the case for
able to find these peptides by mass spectrometry. However, DappuOMTLs which, however, exhibit more divergent se-
these smaller peptides may be processed from their precursor in quences. Thus, both decapod crustacean OKs and OMTLs
locations other than the brain-optic ganglia complex, for clearly lack one amino acid in their sequence compared to
example, gut endocrine cells or ovaries. In some insects, NPF DappuOKs and other arthropod OKs (Suppl. Figures S11C,
type-1 and -2 or smaller peptides, such as the YS/GQVARPR- S11D, Supporting Information).
Famides, even arise from different NPF genes and are post- Periviscerokinins
translationally processed.106,109 DappuNPF1 and DappuNPF2, Periviscerokinins (PVKs) and CAPA-pyrokinins (PKs) were
however, likely arise by alternative splicing of a single gene, a discovered in abdominal perivisceral or perisympathetic neuro-
phenomenon recently reported for silk moth’s BommoNPF1a hemal organs of the American cockroach.114 These peptides are
and BommoNPF1b48 as well. Both DappuNPFs align best with very likely encoded by the same gene as was first described for the
NPFs corresponding to the two NPF subgroups from several capability (capa)-gene in D. melanogaster.115 Its name was coined
insects and single decapod and copepod crustaceans. However, as a reminder of the “abilities” known for related neuropeptides
a recently proposed invertebrate consensus sequence106 is that act as cardio-acceleratory peptides (CAPs). The structure of
largely conserved only in the Daphnia NPF1 sequences (Suppl. this capa-gene is similar to those of many other insects usually
Figure S10B, Supporting Information). encoding 24 PVKs followed by only one PK. While CAPA-
Orcokinin and Orcomyotropin
PVKs typically have a FPRVamide C-terminus, CAPA-PKs
The myoactive tridecapeptide Asn13-orcokinin NFDEIDRSGFG- usually show a much more conserved C-terminus of
FA was first discovered in the crayfish Orconectes limosus8 and further WFGPRLamide.114 FXPRLamides of insects are also encoded
orcokinin (OK) isoforms were identified later in various decapods in a second gene expressing precursors that give rise to peptides
designated as diapause hormones, pheromone-biosynthesis-acti-
and other crustaceans.13 Insect orcokinins first identified in cock-
vating neuropeptides (PBAN116) or PKs.114 Similar PKs were
roaches and locusts110,111 are in most insects and a chelicerate 14aas-
isolated from the shrimp Litopenaeus vannamei117 and later in
long and closely related to crustacean OKs. Most carry the signature
several other decapod crustaceans,59,60,118,119 but PVKs have
sequence NXDEIDR (X being F or L). However, to our knowledge,
never been identified in decapods.
there is no 13aas-long OK as of the typical length and structure of
The Dappu pvk-gene is a 4-exon gene encoding a 188aas long
decapod OKs in insects.
precursor with three different PVKs but no PK (Suppl. Table
Another myoactive peptide orcomyotropin FDAFTTGFa-
S1, Supporting Information). We identified the three Dap-
mide (OMT) was discovered as a potent hindgut stimulatory
puPVKs by fragment analysis (Table 1; Figure 1A,B). Although
factor in the crayfish O. limosus.9 The first precursors for decapod
the general gene structure is similar to those of insect Capa-genes,
crustacean orcokinins contain 7 and 8 copies of four different
only one putative dodecapeptide SNSDERTAVFIP-(RRW) is
orcokinin isoforms, and one copy of the OMT-like undecapep-
encoded close to the C-terminus of the DappuPVK-precursor,
tide (OMTL) FDAFTTGFGHS.112 OMTs and OMTLs have
where one would have expected a pyrokinin. Interestingly, similarly
been identified from several decapods99,113 but a separate OMT-
to the DappuPVK precursor, two slightly different precursors from
precursor has never been found. In insects, OK-precursors
another branchiopod species, the brine shrimp Artemia franciscana,
usually give rise to OMTL peptides in similar positions but only
contain similar ArtfrPVKs but also lack a pyrokinin (ES499501.1,
23 single copies of different OKs.
ES514145.1; Suppl. Figure S12, Supporting Information).
We describe here the structures and products of two orcoki-
nin-genes that occur in the D. pulex genome in different positions Pigment Dispersing Hormone
about 191kb apart from each other on the same scaffold (Suppl. The 2-exon Dappu pdh-gene occurs as a single gene that differs
Table S2, Supporting Information). The Dappu okI-gene en- with regard to length and an intron interrupting the PRP from
codes the isoforms DappuOK1 NLDEIDRSNFGTFA and Dap- the closely related but intron-less gene for the fruit fly pigment
puOK2 NLDEIDRSDFGRFV following two OMTL-related dispersing factor (DromePDF; CG6496; Suppl. Figure S13A,
peptides (DappuOMTL1 and DappuOMTL2) located directly S13B, Supporting Information). The derived precursor structure
upstream (Suppl. Figure S11A, Supporting Information). The of DappuPDH is similar to that of the CarmaPDH and Dro-
Dappu okII-gene is a 3-exon gene encoding DappuOK1 and a mePDF precursors in that the beta-PDH-type octadecapeptide
closely related third isoform DappuOK3 NLDEIDRSDFSRFV sequence of NSELINSLLGLPRFMKVVamide appears at the 30
following a further OMTL-related peptide (DappuOMTL3; end of the DappuPDH-ORF as confirmed here by RT-PCR
Suppl. Figure S11B, Supporting Information). Mass matches (Suppl. Table S2, Supporting Information). DappuPDH is
have confirmed both DappuOK1 and DappuOK2 but not yet identical in D. pulex and D. magna as previously shown by means
DappuOK3 (Table 1; Figure 1D). The sequences of both of mass spectrometry,120 and as also confirmed here (Table 1,
DappuOK precursors containing only two OKs closely resemble Figure 1A). In the two water fleas, it occurs in almost identical
Figure 13. Comparison of the two novel D. pulex and the Drosophila melanogaster proctolin genes and preprohormones. (A) Proctolin genes are similar
with regard to the large introns splitting only the precursor-related peptides. However, the Dappu M4proct-gene and the Drome proct-gene share an
additional first intron splitting the 50 -untranslated regions (M4 Met in position 4; SP or S signal peptide light gray, P proctolin codons dark gray). (B)
Proctolin preprohormones of D. pulex are highly similar to each other (66% aai, 78% scr), but differ considerably in length and lack of polyQ and polyS
stretches from the DromeProct precursor (green). Note the identical proprotein convertase (><) processing site RS; Proctolin peptides are boxed;
∼ and diamond indicate ends of SPs.
complex networks of PDH-ir neurons, some of which are clear potato beetle.126 Interestingly, only the Dappu M4proct-gene
homologues to well-known insect clock neurons.120 The Dap- has a similar 3-exon structure when compared with the Drome
puPDH itself is very different from all known decapod PDHs and proct-gene (CG7105-RA; Figure 13A), although the derived
even from insect PDFs especially with regard to the last three precursor is a lot more similar to that of the DappuProctolin
amino acids and the lack of basic processing aas at the 30 end of precursor than to that of the DromeProctolin precursor (Figure 13B),
the ORF. However, the core structure at the aas 69121 is well suggesting that both Dappu proct- and Dappu M4proct-genes are
conserved (Suppl. Figure S12B, Suppl. Table S1, Supporting the result of a gene duplication.
Information). There was no hint for the existence of an alpha-
PDH type peptide122 or other PDH-isoforms, such as the up to RYamides
three PDH-isoforms in the crayfish Orconectes limosus.123 Many crustaceans and insects contain neuropeptides with the
C-terminal motif RYamide that are reminiscent of but very
Proctolins different from the vertebrate NPY and PPY-like peptides (see
Proctolin was the first insect neuropeptide to be identified, but above). Members of this invertebrate family have been identified
its gene and precursor structure in insects (D. melanogaster) was by mass spectrometry from the pericardial organs of several
one of the latest to be unraveled.124,125 Here, we describe two decapod crustaceans,59,127 but their precursor structures are as
different proctolin genes in the D. pulex genome. The newly yet unknown. Stemmler et al. (2007)128 corrected a widely
annotated 2-exon Dappu proct-gene and the 3-exon Dappu distributed but previously in several decapods misidentified
Met4-proct- gene are strikingly different and occur on different RYamide family member to be pQGFYSQRYamide. In insects,
scaffolds (Suppl. Table S1, Supporting Information; Figure 13A). a large diversity of RYamides has recently been discovered in the
However, the derived precursors resemble proctolin precursors jewel wasp N. vitripennis and many other insects with annotated
of two different Drosophila species124 (Figure 13B) and in both genomes.80 However, their C-terminal consensus sequence
cases the SP is followed by a proctolin (RYLPT or RYLMT; FFxxxRYamide is somewhat different from the above-mentioned
Table 1). We have identified both proctolin isoforms by frag- RYamide. The insect RYamide precursors contain at least one
mentation sequencing. DappuMet4-proctolin is a novel proctolin short and one long RYamide, and some of them have an
variant in arthropods. This is surprising since proctolin is an additional elongated RYamide or RFamide.
extremely conserved arthropod peptide. In insects, there is only The Dappu rya-gene gives rise to a precursor entirely novel for
one exception known as Ala1-proctolin from the Colorado crustaceans that liberates a short and a long typical RYamide, and
SIFamide always occurs directly after the SP followed by a so-

called joining peptide.130 Isoforms by one amino acid shorter
have been predicted from ESTs after BLAST searches for
D. pulex and the pea aphid Acyrthosiphon pisum only.130
The Dappu sifa-gene of D. pulex is a 2-exon gene encoding
a 73aas long precursor with a single undecapeptide
TRKLPFNGSIFa. We have confirmed this unique peptide by
MALDI-TOF (Table 1, Figure 1C). DappuSIFamide diverges
significantly in two positions from the signature mentioned
above. The lack of Tyr2 and substitution of Pro5 (Leu4 in
DappuSIFa) have not been described for any other arthropod
species before. The overall precursor structure is, however, very
similar to SIFamide precursors from insects, crustaceans and a
chelicerate (Suppl. Figure S15, Supporting Information). These
structural similarities and especially the occurrence of two
cysteines in the joining peptides or PRPs have tempted authors
to speculate on a common origin of AKH, corazonin and
SIFamide genes.56,130
Figure 14. Alignment of DappuSNPF with other arthropod sNPFs. Short NPF
The novel undecapeptide DappuSNPF is clearly closer related to most Members of the short neuropeptide F (sNPF)-family, that is,
insect sNPFs than to all invertebrate sNPF-like peptides, but still differs hexa- to undecapeptides carrying a C-terminus XRXRFamide
from all hitherto known sNPF sequences. Note that even the unusual (X being a variable residue but usually Leu109) as their signature,
dibasic flanking sequences (RK, RR) used for precursor processing are have been discovered in the Colorado potato beetle135 and later
highly conserved. To date other crustacean and Limulus sNPF pre- identified from many insects.107,109 The first crustacean members
cursors are not yet known. (Insect (green) sNPFs from: pea aphid were discovered in the shrimp M. rosenbergii136 and later in
Acyrthosiphon pisum FP925011, mosquito A. gambiae A0SIF1.1, honey-
several decapod species from four different taxa.13,107
bee A. mellifera HP467766.1, silkworm B. mori NM_001134257,
D. melanogaster CG13968, Colorado potato beetle Leptinotarsa decemlineata The 4-exon Dappu snpf-gene encodes a single short neuro-
sNPF1,135 L. migratoria P86444, P. americana head peptide peptide F (Dappu-sNPF), the undecapeptide SDRSPSLRL-
(PeramHP156), red flour beetle T. castaneum;57 from the chelicerate tick RFamide, which we confirmed by fragment analysis (Table 1,
Ixodes scapularis XM_002408268.1 (orange); from the Limulus polyphemus Figure 1A,C), and likely four short peptides flanked by putative
head peptide157 (orange); crustacean (dark blue) sNPFs from: C. maenas,59 monobasic cleavage sites. Characteristically, the last phase-1
H. americanus,118 shrimp Penaeus monodon FLP6,132 PenmoVal2-FLP6.158. intron 3 splits the coding region between Dappu-sNPF18 and
Dappu-sNPF.911 This is unique among arthropod snpf-genes.
an elongated 22-mer with the RFamide C-terminus (Suppl. Mass spectrometric analysis revealed an Asp in position 2, which
Table S1; Figure S14A, Supporting Information). Another is another unique feature among all arthropod sNPFs. Dappu-
branchiopod species, Artemia parthenogenetica, has a similar gene sNPF is much more similar to sNPFs from diverse insect groups
that codes for two more small PRPs upstream of the first RYamide than to any decapod crustacean sNPF or sNPF-like peptide
(Figure S14A, Supporting Information). We have confirmed (Figure 14). In fact, the only residues identical to crustacean
the DappuRYa1 pQTFFTNGRYamide and DappuRYa31727 sNPFs reside in the C-terminal RxPxLRLRFamide consensus
by fragmentation sequencing (Figure 4), and the longer sequence (with the exception of Asp1-MacroFLP5132), whereas
27-mer DappuRYa3 by mass match (Table 1, Figure 1A, 1B). identities to insect peptides amount to 810 out of 11aas and
Alignments of decapod, branchiopod (Daphnia and Artemia), even extend further to the unusual processing sites (RK, RR)
and insect RYamide sequences show that the DappuRYamides typically flanking the sNPFs at both sides. The other peptides
are somewhat more similar to the insect than to the decapod encoded by the Dappu-sNPF precursor have no resemblance
sequences as shown in Suppl. Figures S14BE (Supporting with any other known arthropod peptide.
Information).
Sulfakinins
SIFamide Sulfakinins are considered the arthropod homologues of verte-
Members of a highly conserved dodecapeptide family brate gastrin/cholecystokinin (G/CCK)-related peptides and are
of SIFamides were first isolated as the myotropic peptide multifunctional regulators of feeding behavior. Discovered in cock-
NeobuLFamide from the flesh fly Neobellieria bullata.129 They roaches as leucosulfakinins I and II, well-conserved sulfakinins with a
are ubiquitous in insects and characteristically show a motif characteristic heptapeptide structure containing a sulphated tyrosine
XYRKPPFNGSIFamide (X1 usually being R, G, T),130 but only XY(SO3H)GHMRFamide (X = D or E) are known in several
for D. melanogaster SIFamide a function in the control of arthropod groups.137,138 The Tyr-sulfation is usually considered
reproductive behavior is known.131 Most crustaceans express vital for full bioactivity, although cardio-stimulatory activity has been
Gly1-SIFamide, first identified in the tiger prawn P. monodon132 described for nonsulphated sulfakinins.139
but a neuromodulatory isoform Val1-SIFamide was recently In the D. pulex genome we found a single 4-exon Dappu
identified in lobsters.13 In crayfish133 and lobsters,134 cloned sk-gene that gives rise to a precursor with two sulfakinins, Dap-
SIFamide precursors showed only single copies of either puSK1 and DappuSK2, in tandem arrangement (Suppl. Table S1,
isoform.134 The relatively short precursors show similarities in Supporting Information). We have confirmed the predicted
overall structure to those of insects, among which also alternative exon/intron structure of the Dappu sk-gene by RT-PCR
splice forms of SIFamide precursors exist. Characteristically, (Suppl. Table S2, Supporting Information) and have sequenced
DappuSK1, its noncyclized Gln1-isoform (relative amounts: were sequenced by MS/MS. From the 45 derived precursors, a
30/70%, respectively), and DappuSK2 by mass spectrometry dozen critical precursors were confirmed by RT-PCR including
(Table 1, Figures 1B, 9C). We did not find any tyrosine-sulfated four cases derived from two single genes that give rise to two
form, although sulfinator-based analysis predicted a sulfation for precursors each by alternative splicing processes (ITP/ITPL,
Tyr5 of DappuSK2 (E-value 0.097). DappuSK1 is N-terminally NPF1/2; Suppl. Table S2, Supporting Information).
blocked by a pyro-glutamate residue and shows a unique Tyr-
Novel Neuropeptide and Protein Hormone Genes and Gene
amide C-terminus not known so far for sulfakinins from any
Products
arthropod species, whereas DappuSK2 follows the canonical
Although increasing numbers of putative neuropeptide precursor
signature. Apart from the C-terminal Tyr-amide, the DappuSK1
sequences are nowadays derived from sequenced genomes and
sequence differs by two aas (Pro2, Asp4) from pQFDEYGHMR-
ESTs, peptide identification studies remain indispensible to reveal
Famide, the identical SK1 in likely all decapod SK1-SK2 pre-
the physiologically relevant primary structures. Our mass spectro-
cursor tandems. However, it differs by one aa (Pro2) only from
metric profiling from D. pulex brain extracts and directly from brain
the SK1s of cockroaches. DappuSK2 is not only shorter than all
tissues enabled us to newly identify and/or confirm bioinformatically
decapod SK2s but also contains the canonical -HMRFamide
predicted precursors and peptides (Table 1, Suppl. Table S1, S3,
signature found in all insect SK2s but not in any decapod SK2
Supporting Information); we also confirmed a number of difficult to
(-HLRFamides). DappuSK2 also resembles most closely all dip-
annotate gene structures by RT-PCR (Suppl. Table S2, Supporting
teran SK1s and nearly all SK2 sequences with regard to the
Information). As a result, we can state that all the peptides detected in
nonapeptide signature sequence beginning with an identical Phe2
brain samples (Table 1) are true neuropeptides. Nevertheless, several
in DappuSK2 and a Phe in the same position of all insect SK2s.
peptides may just not have been detected, because they were either
All decapod SK2s have instead a Tyr in that position (Suppl.
present in too low amounts, that is, under the detection limit,
Figure S16, Supporting Information).
detectable only at different locations in the body, or present only at
Tachykinin-Related Peptides certain developmental stages, as is well-known, for example, for insect
Members of the invertebrate tachykinin-related peptide ETHs. Incongruences between our and some previously EST-
(TKRP) family are common to all arthropods, some other predicted precursor sequences37 may be due to known hybrid
ecdysozoans and mollusks and share similarity with vertebrate formations of D. pulex and D. pulicaria being responsible for
tachykinins and neurokinins. Arthropod TKRPs are character- erroneous EST resources (John Colbourne, Pers. comm.).
ized by a C-terminal FX1GX2Ramide motif (X1,X2 are variable We discovered, confirmed and partly identified by mass
aas140). In crustaceans, the nonapeptide CabTRP APSGFLGM- spectrometry a number of novel neuropeptide and protein
Ramide was the first to be discovered in the CNS of the crab C. hormone genes and their distinct, even post-translationally
borealis;141 later on slightly different isoforms of the same length modified products in D. pulex. Among these are the allatotropin,
were identified in the CNS of several other decapod and isopod putative diuretic hormones such as a calcitonin-like DappuDH31
species and recently also as TPSGFLGMRamide in midguts of and a CRF-related DappuDH52, two novel splice forms of
several decapods.142,143 vertebrate NPY-related DappuNPF, three ASTC-type peptides
The D. pulex genome contains a single 3-exon Dappu tkrp- derived from different genes, the two proctolin variants RYLPT
gene encoding a 248bp long precursor containing single copies of and RYLMT, an Ala7-CCAP isoform, an oxytocin-like inotocin,
three different DappuTKRPs on the second exon. We have four insulin/IGF/relaxin-superfamily peptides, one being a clo-
confirmed this gene structure partially by RT-PCR and identified sely insulin-related peptide (DappuIRP2), the others being more
all DappuTKRPs by mass spectrometry (Table 1, Suppl. Table IGF-like peptides, and four ITP or ITP-like peptides. Entirely
S2, Supporting Information, Figure 1B,C). The DappuTKRPs new to crustaceans were also the enigmatic CCHamide, two
are undecapeptides that share the C-terminal signature sequence eclosion hormone variants, two ETHs, glycoprotein hormone
but their N-terminal sequences neither resemble any of the GPA2/GPB5, RYamides, a novel sNPF, and PVKs.
decapod TKRP nonapeptides nor any of the insect TKRP nona-
Gene Expansions by Novel Alternative Splice Forms
or decapeptides, except for the blowfly CalvoTKII undecapep-
Alternative splicing is a well-known process to functionally
tide and N-terminally extended cockroach, locust and fly TKRP-
and structurally diversify many eukaryote gene products from
isoforms13,140,144 (Suppl. Figure S17, Supporting Information).
one and the same gene. Small and large peptides such as the ATs
As in the Dappu tkrp-gene and the irregular 24 exon tkrp-genes
and ITP/ITPLs as well-known examples are differentially ex-
of insects, a maximum of two exons only carry the entire TKRP-
pressed in different tissues as products of alternative splicing in
ORFs. Interestingly, however, a comparison of the TKRP-pre-
insects and the latter also in crustaceans.93,145,146 Other authors
cursors reveals that the DappuTKRP precursor appears to be an
have found for instance products of diuretic hormone (dh)- and
intermediate between insect TKRP-precursors, which are pro-
capa-pvk/pk-genes to be alternatively spliced in complex
cessed into more than three (max. 13) diverse single copy
ways.48,57 We did not find any clear-cut evidence for alternative
TKRPs, and decapod TKRP-precursors, which give rise to
splice forms of DappuAT, the two novel types of Daphnia DHs,
several copies of a single TKRP sequence or only one additional
neither the CRF-like DappuDH52 nor the calcitonin-like Dap-
different TKRP isoform as in H. americanus (Suppl. Figure S18,
puDH31, for which splice forms are known from some insects.
Supporting Information).
However, DappuITP and DappuITPL clearly occur as distinct
splice forms in a way similar to those derived from a single crab
’ DISCUSSION AND CONCLUSIONS CHH/ITP-gene92 and putatively similar genes in other crusta-
We present here the first global analysis of neuropeptides and ceans and insects.93 Recently, similar splice forms of ITP/ITPL-
protein hormones present in a crustacean species. We annotated type peptides have been identified in the closely related bran-
43 genes coding for 73 neuropeptides. From these, 40 neuropep- chiopod species Daphnia magna and other crustaceans.147 As a
tides were confirmed by mass matches (MALDI-TOF) and 30 novel example for alternative splice forms, we found that the
Dappu npf-gene leads to the DappuNPF1 and the DappuNPF2 contradictory examples of peptides and precursors more closely
splice forms, as evidenced by the RT-PCR results. related to decapod peptides (see below).
Similarity trait expressions allowing to put the D. pulex
Gene Expansions by Gene Duplication: Gene Clustering and peptidome closer to insect than crustacean peptidomes are found
Split Messages in a number of D. pulex peptides such as the very clearly insect
A striking feature of many genes is the clustering of closely AKH-like DappuAKH, DappuASTBs, DappuASTC2, DappuEH,
related genes. This is often indicative of gene duplication events DappuIRPs, DappuITP/ITPLs, DappuMS, DappuOKs,
during evolution, which appear to be particularly common in the DappuOMTLs, DappuRYamides, Dappu-sNPF, and DappuSKs.
D. pulex genome.15 Initially, we expected to find such gene The first hint clearly suggesting a closer relationship to corre-
clusters for CHH-like ITP/ITPL-genes since this phenomenon sponding insect rather than decapod peptide families, came
is well established for a shrimp CHH-gene family,14 but we found from the notion of an obvious absence of type-II MIH/VIH-like
only one typical CHH-like Dappu itp-gene and two other quite peptides and from the observed high N- and C-terminal
different ITP-like genes each producing novel N-terminally sequence similarities of DappuITP/ITPL-like peptides to insect
elongated ITPs, but all three genes occurring on different ITP/ITPLs. The occurrence of MIH/MOIH/VIH-like type-II
scaffolds. Interestingly, for four D. pulex peptide families we peptides only in decapod crustaceans and their total absence
found clusters of closely related paired peptide genes, some right from insects has previously been interpreted as a novel acquisi-
next to each other but with mostly very different structures. The tion related to different modes of molt control in decapods and
paired clustered genes of ASTC2/ASTC1-peptides, long/short insects.2 Further support derives from a recent extensive
EHL/EH, and the cystine-knot protein hormones bursicon and molecular phylogenetic analysis of these peptide families, which
glycoprotein GPA2/GPB5 are such examples (Figure 5). The showed that insect and Daphnia ITP/ITPLs can clearly be
“closest relatives” among these paired clusters were found in the grouped together in a novel type-III subgroup, and led to the
cases of the genes for the bursicons and the glycoprotein GPA2/ assumption of several gene duplications by intron insertions into
GPB5, which in both protein hormone families form dimers as a an ancestral 2-exon gene.147 However, cloning and expression
prerequisite for bioactivity. In several cases, a tendency to expand analyses are needed to confirm this attractive hypothesis for the
genes by gene duplication was notable even in the rather Dappu itpn-gene as a comparable 2-exon gene candidate. On the
unexpected cases of proctolin and Met4-proctolin, eclosion other hand, the Dappu itpln-gene is not a typical 4-exon type-I
hormones, orcokinins, and ASTC1 to ASTC3, and perhaps gene like the Dappu itp-gene, since it appears to have lost the
DappuIRP3 and DappuIRP4. In insects and nematodes, it canonical half of the short CHH/ITP-splice form normally
appears to be quite normal that insulin genes have multiplied. encoded at the beginning of its fourth exon but shows instead
However, there is certainly one single distinct DappuIRP2 that only the half of the long ITPL-splice form encoded as usual in the
apparently shows all structural characteristics of insulin, whereas third exon. Thus, with regard to gene and precursor structures, the
the other three DappuIRPs appear to be more IGF-like peptides. two Daphnia itpln- and itpn-genes rather resemble more rapidly
We noticed for several D. pulex neuropeptide genes that the evolved type-II MIH/VIH-genes only in that they still have
neuropeptide messages were split by introns into splicable exons “retained” most of the typical signatures of the type-I CHH/
but without any regularity regarding the introns’ phase. Such split ITP-gene, precursor and peptide structures. These genes and their
messages were found not surprisingly for CCAP and CCHamide, extended ITP-related products may thus represent evolutionary
since this feature is known from several insect ccap- and ccha- intermediates between those of CHHs/ITPs and MIHs/VIHs.
genes as discussed above, but occurs obviously as a unique feature Sequence constraints and distinctions were recognized for
also for corazonin in the Dappu crz-gene, for the ASTA5 in DappuIRP2 as showing one additional aa between the cystine-
the Dappu astA-gene, and the DappuIRP B- and -C chains in the bridges of the A-chain and four aas lacking between the cystine-
Dappu irp2-gene, and the short NPF in the Dappu snpf-gene. bridge of the B-chain as is typical for insect IRPs but not found
Whether this happened just by chance during evolution or in decapod crustaceans (Suppl. Figure S6, Supporting In-
together with known massive events of intron gain in Daphnia formation). Moreover, clear-cut differences in peptide length
species148 remains to be clarified. exist for DappuOKs and DappuSKs, as e.g. 14aas long OKs in
insect and Daphnia OKs vs strictly 13aas long OKs in decapod
D. pulex Peptidome Origins Closer to Insects or Intermedi- crustaceans only, and the occurrence of HMRFamide signatures
ate between Insects and Decapod Crustaceans? in insect and Daphnia vs HFRFamides in decapod sulfakinins
Repeatedly, our thorough analysis of the D. pulex peptide and (Suppl. Figures S11, S16, Supporting Information). A similar
protein hormone gene organizations and gene products revealed structure is seen in the clearly insect-like Val3-DappuMS vs
striking similarities and closer relationships to insect rather than strictly Leu3-myosuppressins found in all decapod crustaceans.
decapod crustacean peptidome members. One may argue that Furthermore, DappuRYamides with their insect-like heptamer
this outcome might have been biased by the preferred use of compared with the typical hexamer signature of decapod RYa-
“insect queries” to detect similar crustacean genes. However, mides, the Dappu-sNPF being clearly more similar to all insect
even if this were the case and several peptide genes still remain to sNPFs compared with the very different decapod sNPFs, are
be discovered, the closer examination of available gene struc- other examples supporting our hypothesis. High sequence
tures, precursors and peptide sequences allowing for stringent identity of peptides may result from independent evolutionary
analyses supported by quantified alignments strongly suggests a processes due to shared evolutionary constraints limiting se-
closer relationship of D. pulex and insect rather than decapod quence diversifications. However, not only peptide sequences,
crustacean peptidomes. In addition, there were several hints but also gene- and precursor structures in D. pulex often
from, for example, D. pulex peptide precursors indicating more represented organizations “intermediate” between those of dec-
“intermediate” structural characteristics combining traits from apods and insects as became evident for several neuropeptide
one or the other compared systematic subgroup, but also a few genes. Characters such as (i) a different peptide length (lacking
Tyr2) and an exchanged Leu4 in the sequence of DappuSIFamide limiting for reasonably detailed examinations. To investigate
but otherwise similar precursor structures (Suppl. Figure S15, whether the observed closer relationship with insect neuropep-
Supporting Information), (ii) again different length of three tidomes anticipates further surprises in unraveling cladoceran
DappuTKRPs undecapeptides when compared with highly regulatory principles, which perhaps again resemble more those
diversified shorter insect TKRP and the single but multiplied of insects than those established for decapods, will be major
TKRPs on the respective decapod crustacean precursors (Suppl. challenges for the future but exciting. Another promising per-
Figure S18, Supporting Information), (iii) the largely expanded spective is the use of the well-annotated D. pulex genomic
diversity in ASTA-peptides in decapods and insects contrary to information for deletion experiments by RNA-interference to
the six DappuASTAs, and (iv) comparable insect CAPA-PVK/ silence single or several genes as shown recently for D. magna151
PK precursors but lacking PKs in the DappuPVK-precursor and analyze changes in gene expression and behavioral pheno-
compared with putatively lacking PVKs in decapod CAPA-PK- types even genome-wide.
precursors are examples for “intermediate” manifestations of
D. pulex peptide gene structures and products, although decapod ’ ASSOCIATED CONTENT
PK-precursors have yet to be unraveled. At last, the identification
of three different OK-precursors in lobster113 has indicated the bS Supporting Information
occurrence of up to three different ok-genes in decapods. Thus, Supplementary Tables S1, S2 and S3, and Supplementary
the presence of two copies of Dappu ok-genes compared to only Figures S1S18. This material is available free of charge via the
one copy of ok-gene in insects (or even its likely loss in dipterans Internet at http://pubs.acs.org.
and moth111) may be a good example for an intermediate
outcome of gene expansions in D. pulex. Although clearly closely ’ AUTHOR INFORMATION
related to insects, several novel neuropeptides and protein
hormones of D. pulex cannot be used for the comparison and Corresponding Author
discussion of their insect vs decapod relationships, since they are *Heinrich Dircksen, Stockholm University, Dept. Zoology, Svante
entirely new to crustaceans and have so far not even been found Arrhenius v€ag 18A, S-10691 Stockholm, Sweden; Tel. +46-
in any decapod crustacean. Only a few analyzed D. pulex peptide 8-164076, Fax +46-8-167715, e-mail: dircksen@zoologi.su.se.
and protein hormone gene products appeared to contradict our
hypothesis as in the cases of DappuASTC1 and DappuASTC3, ’ ACKNOWLEDGMENT
DappuBursA and B, and Dappu neuroparsin hormones in that
the former peptides or their precursors were identical or slightly We thank the Carl Tryggers Foundation, Stockholm, Sweden
closer related to their decapod than to their insect counterparts in (to J.S. and H.D.), the Faculty of Sciences, Stockholm University
our quantified alignments. (to H.D.), the Fund for Scientific Research Flanders, Belgium
These findings are consistent with a close phylogenetic (F.W.O.-Vlaanderen, to P.V., J.H., K.P.), the German Research
relationship of branchiopods and insects, which has been inferred Foundation (DFG to R.P.), the Danish Research Council for
from several molecular phylogenetic studies using ribosomal and Nature and Universe (to C.J.P.G. and F.H.), and the Novo Nordisk
mitochondrial sequences,149 although the relationships among Foundation (to C.J.P.G. and F.H.) for financial support and J€org
Pancrustacea are far from being finally resolved.150 Altogether Kahnt (MPI Marburg, Germany) for help during mass spectro-
our results lend further support to a previous notion from very metric analysis. We are part of the Daphnia Genome Sequencing
different gene analyses showing that branchiopods are much Consortium and thank all the people that have contributed to
closer related to insects than to decapod crustaceans and that the this effort.
evolution of land-living insects went through freshwater habitats
populated by branchiopods as last common ancestors of hex- ’ REFERENCES
apods and crustaceans.16 The data presented here thus provide (1) Fernlund, P.; Josefsson, L. Chromactivating hormones of Pan-
an important new source of information on functional aspects of dalus borealis. Isolation and purification of the red-pigment concentrat-
neuropeptidome evolution. ing hormone. Biochim. Biophys. Acta 1968, 158, 262–273.
(2) Chen, S. H.; Lin, C. Y.; Kuo, C. M. In silico analysis of crustacean
Aspects of Neuropeptide Functions in Branchiopods hyperglycemic hormone family. Mar. Biotechnol. 2005, 7 (3), 193–206.
(3) B€ ocking, D.; Dircksen, H.; Keller, R. The crustacean neuropep-
For none of the peptides described here, any clear-cut func-
tides of the CHH/MIH/GIH family: Structures and biological activities.
tional context is so far known in D. pulex. However, from the In The Crustacean Nervous System; Wiese, K., Ed.; Springer: Berlin,
broad knowledge about insect and decapod crustacean physiol- Heidelberg, 2002; pp 8497.
ogies, one can anticipate that concept-building experimentation (4) Mangerich, S.; Keller, R.; Dircksen, H.; Rao, K. R.; Riehm, J. P.
is now greatly facilitated by the availability of primary structures Immunocytochemical localization of pigment-dispersing hormone
of key players in many physiological processes. For instance, (PDH) and its coexistence with FMRFamide-immunoreactive material
nothing is known about molt control mechanisms in cladocer- in the eyestalks of the decapod crustaceans Carcinus maenas and
ans; nevertheless the existence of almost all putative key player Orconectes limosus. Cell Tissue Res. 1987, 250, 365–375.
peptides and peptide genes similar to those of insects and (5) Orchard, I.; Belanger, J. H.; Lange, A. B. Proctolin: a review with
decapods (except for MIHs) as shown here points to functional emphasis on insects. J. Neurobiol. 1989, 20 (5), 470–496.
(6) Dircksen, H., Conserved crustacean cardioactive peptide
similarities. This knowledge will certainly spawn interesting
(CCAP) neuronal networks and functions in arthropod evolution. In
physiological and phylogenetic works with regard to similar if Recent advances in arthropod endocrinology; Coast, G. M., Webster, S. G.,
not homologous genomic, precursor structures and functional- Eds.; Cambridge University Press: Cambridge, 1998; Vol. 65, pp 302333.
ities of derived peptides among all Ecdysozoans. The fast molt (7) Mercier, A. J.; Friedrich, R.; Boldt, M. Physiological functions of
cycles may facilitate investigations into their regulation, but, on FMRFamide-like peptides (FLPs) in crustaceans. Microsc. Res. Tech.
the other hand, the very rapid ecdysis-behaviors may become 2003, 60 (3), 313–324.

(8) Stangier, J.; Hilbich, C.; Burdzik, S.; Keller, R. Orcokinin: a novel (25) Predel, R.; Kellner, R.; Rapus, J.; G€ade, G. Allatostatins from the
myotropic peptide from the nervous system of the crayfish, Orconectes retrocerebral complex and antennal pulsatile organ of the American
limosus. Peptides 1992, 13 (5), 859–864. cockroach: structural elucidation aided by matrix-assisted laser desorp-
(9) Dircksen, H.; Burdzik, S.; Sauter, A.; Keller, R. Two orcokinins tion/ionization-time-of-flight mass spectrometry. Regul. Pept. 1999, 82
and the novel octapeptide orcomyotropin in the hindgut of the crayfish (13), 81–89.
Orconectes limosus: Identified myostimulatory neuropeptides originating (26) Yasuda-Kamatani, Y.; Yasuda, A. Characteristic expression
together in neurones of the terminal abdominal ganglion. J. Exp. Biol. patterns of allatostatin-like peptide, FMRFamide-related peptide, orco-
2000, 203 (18), 2807–2818. kinin, tachykinin-related peptide, and SIFamide in the olfactory system
(10) Duve, H.; Johnsen, A. H.; Maestro, J. L.; Scott, A. G.; Jaros, of crayfish Procambarus clarkii. J. Comp. Neurol. 2006, 496 (1), 135–147.
P. P.; Thorpe, A. Isolation and identification of multiple neuropeptides (27) Yin, G.-L.; Yang, J.-S.; Cao, J.-X.; Yang, W.-J. Molecular cloning
of the allatostatin superfamily in the shore crab Carcinus maenas. Eur. J. and characterization of FGLamide allatostatin gene from the prawn,
Biochem. 1997, 250 (3), 727–734. Macrobrachium rosenbergii. Peptides 2006, 27 (6), 1241–1250.
(11) Dircksen, H.; Skiebe, P.; Abel, B.; Agricola, H.; Buchner, K.; (28) Christie, A. E.; Sousa, G. L.; Rus, S.; Smith, C. M.; Towle, D. W.;
Muren, J. E.; N€assel, D. R. Structure, distribution, and biological activity Hartline, D. K.; Dickinson, P. S. Identification of A-type allatostatins
of novel members of the allatostatin family in the crayfish Orconectes possessing -YXFGI/Vamide carboxy-termini from the nervous system of
limosus. Peptides 1999, 20 (6), 695–712. the copepod crustacean Calanus finmarchicus. Gen. Comp. Endocrinol.
(12) Duve, H.; Johnsen, A. H.; Scott, A. G.; Thorpe, A. Allatostatins 2008, 155 (3), 526–533.
of the tiger prawn, Penaeus monodon (Crustacea: Penaeidea). Peptides (29) Belles, X.; Graham, L. A.; Bendena, W. G.; Ding, Q.; Edwards,
2002, 23 (6), 1039–1051. J. P.; Weaver, R. J.; Tobe, S. S. The molecular evolution of the allatostatin
(13) Christie, A. E.; Stemmler, E. A.; Dickinson, P. S. Crustacean precursor in cockroaches. Peptides 1999, 20 (1), 11–22.
neuropeptides. Cell. Mol. Life Sci. 2010, 67 (24), 4135–4169. (30) Hoffmann, K. H.; Meyering-Vos, M.; Lorenz, M. W. Allatosta-
(14) Gu, P. L.; Chan, S. M. The shrimp hyperglycemic hormone-like tins and allatotropins: Is the regulation of corpora allata activity their
neuropeptide is encoded by multiple copies of genes arranged in a primary function? Eur. J. Entomol. 1999, 96 (3), 255–266.
cluster. FEBS Lett. 1998, 441 (3), 397–403. (31) Fu, Q.; Tang, L. S.; Marder, E.; Li, L. Mass spectrometric
(15) Colbourne, J. K.; Pfrender, M. E.; Gilbert, D.; Thomas, W. K.; characterization and physiological actions of VPNDWAHFRGSW-
Tucker, A.; Oakley, T. H.; Tokishita, S.; Aerts, A.; Arnold, G. J.; Basu, amide, a novel B type allatostatin in the crab, Cancer borealis. J. Neurochem.
M. K.; Bauer, D. J.; Caceres, C. E.; Carmel, L.; Casola, C.; Choi, J.-H.; 2007, 101 (4), 1099–1107.
Detter, J. C.; Dong, Q.; Dusheyko, S.; Eads, B. D.; Fr€ohlich, T.; Geiler- (32) Weaver, R. J.; Audsley, N. Neuropeptide regulators of juvenile
Samerotte, K. A.; Gerlach, D.; Hatcher, P.; Jogdeo, S.; Krijgsveld, J.; hormone synthesis: structures, functions, distribution, and unanswered
Kriventseva, E. V.; Kultz, D.; Laforsch, C.; Lindquist, E.; Lopez, J.; questions. Ann. N.Y. Acad. Sci. 2009, 1163, 316–329.
Manak, J. R.; Muller, J.; Pangilinan, J.; Patwardhan, R. P.; Pitluck, S.; (33) Veenstra, J. A.; Allatostatin, C and its paralog allatostatin double
Pritham, E. J.; Rechtsteiner, A.; Rho, M.; Rogozin, I. B.; Sakarya, O.; C: the arthropod somatostatins. Insect Biochem. Mol. Biol. 2009, 39 (3),
Salamov, A.; Schaack, S.; Shapiro, H.; Shiga, Y.; Skalitzky, C.; Smith, Z.; 161–170.
Souvorov, A.; Sung, W.; Tang, Z.; Tsuchiya, D.; Tu, H.; Vos, H.; Wang, (34) Dickinson, P. S.; Wiwatpanit, T.; Gabranski, E. R.; Ackerman,
M.; Wolf, Y. I.; Yamagata, H.; Yamada, T.; Ye, Y.; Shaw, J. R.; Andrews, R. J.; Stevens, J. S.; Cashman, C. R.; Stemmler, E. A.; Christie, A. E.
J.; Crease, T. J.; Tang, H.; Lucas, S. M.; Robertson, H. M.; Bork, P.; Identification of SYWKQCAFNAVSCFamide: a broadly conserved
Koonin, E. V.; Zdobnov, E. M.; Grigoriev, I. V.; Lynch, M.; Boore, J. L. crustacean C-type allatostatin-like peptide with both neuromodulatory
The ecoresponsive genome of Daphnia pulex. Science 2011, 331 (6017), and cardioactive properties. J. Exp. Biol. 2009, 212, 1140–1152.
555–561. (35) Ma, M.; Szabo, T. M.; Jia, C.; Marder, E.; Li, L. Mass spectro-
(16) Glenner, H.; Thomsen, P. F.; Hebsgaard, M. B.; Sorensen, metric characterization and physiological actions of novel crustacean
M. V.; Willerslev, E. Evolution. The origin of insects. Science 2006, 314 C-type allatostatins. Peptides 2009, 30 (9), 1660–1668.
(5807), 1883–1884. (36) Stemmler, E. A.; Bruns, E. A.; Cashman, C. R.; Dickinson, P. S.;
(17) Van Harreveld, A. A physiological solution for freshwater Christie, A. E. Molecular and mass spectral identification of the broadly
crustaceans. Proc. Soc. Exp. Biol. Med. 1936, 34, 428–432. conserved decapod crustacean neuropeptide pQIRYHQCYFNPISCF:
(18) Bendtsen, J. D.; Nielsen, H.; von Heijne, G.; Brunak, S. the first PISCF-allatostatin (Manduca sexta- or C-type allatostatin) from
Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 2004, a non-insect. Gen. Comp. Endocrinol. 2010, 165 (1), 1–10.
340, 783–795. (37) Gard, A. L.; Lenz, P. H.; Shaw, J. R.; Christie, A. E. Identification
(19) Veenstra, J. A. Mono- and dibasic proteolytic cleavage sites in of putative peptide paracrines/hormones in the water flea Daphnia pulex
insect neuroendocrine peptide precursors. Arch. Insect Biochem. Physiol. (Crustacea; Branchiopoda; Cladocera) using transcriptomics and
2000, 43 (2), 49–63. immunohistochemistry. Gen. Comp. Endocrinol. 2009, 160 (3), 271–
(20) Southey, B. R.; Amare, A.; Zimmerman, T. A.; Rodriguez-Zas, 287.
S. L.; Sweedler, J. V. NeuroPred: a tool to predict cleavage sites in (38) Elekonich, M. M.; Horodyski, F. M. Insect allatotropins belong
neuropeptide precursors and provide the masses of the resulting to a family of structurally-related myoactive peptides present in several
peptides. Nucleic Acids Res. 2006, 34 (Web Server issue), W267–272. invertebrate phyla. Peptides 2003, 24 (10), 1623–1632.
(21) Gaus, G.; Kleinholz, L. H.; Kegel, G.; Keller, R. Isolation and (39) Lee, K. Y.; Chamberlin, M. E.; Horodyski, F. M. Biological
characterization of of red-pigment-concentrating hormone from six activity of Manduca sexta allatotropin-like peptides, predicted products
crustacean species. J. Comp. Physiol. B 1990, 160, 373–379. of tissue-specific and developmentally regulated alternatively spliced
(22) Martinez-Perez, F.; Valdes, J.; Zinker, S.; Arechiga, H. The mRNAs. Peptides 2002, 23 (11), 1933–1941.
genomic organization of the open reading frame of the red pigment (40) Abdel-latief, M.; Meyering-Vos, M.; Hoffmann, K. H. Molecu-
concentrating hormone gene in the blue crab Callinectes sapidus. Peptides lar characterisation of cDNAs from the fall armyworm Spodoptera
2002, 23 (4), 781–786. frugiperda encoding Manduca sexta allatotropin and allatostatin prepro-
(23) G€ade, G.; Hoffmann, K. H.; Spring, J. H. Hormonal regulation hormone peptides. Insect Biochem. Mol. Biol. 2003, 33 (5), 467–476.
in insects: facts, gaps, and future directions. Physiol. Rev. 1997, 77 (4), (41) Paemen, L.; Tips, A.; Schoofs, L.; Proost, P.; Van Damme, J.; De
963–1032. Loof, A. Lom-AG-myotropin: a novel myotropic peptide from the male
(24) Marco, H. G.; G€ade, G. Biological activity of the predicted red accessory glands of Locusta migratoria. Peptides 1991, 12 (1), 7–10.
pigment-concentrating hormone of Daphnia pulex in a crustacean and (42) Honegger, H. W.; Dewey, E. M.; Ewer, J. Bursicon, the tanning
an insect. Gen. Comp. Endocrinol. 2010, 166 (1), 104–110. hormone of insects: recent advances following the discovery of its

molecular identity. J. Comp. Physiol., A: Neuroethol. Sens. Neural. Behav. L. Characterization of the Carcinus maenas neuropeptidome by mass
Physiol. 2008, 194 (12), 989–1005. spectrometry and functional genomics. Gen. Comp. Endocrinol. 2009,
(43) Van Loy, T.; Van Hiel, M. B.; Vandersmissen, H. P.; Poels, J.; 161 (3), 320–334.
Mendive, F.; Vassart, G.; Vanden Broeck, J. Evolutionary conservation (60) Ma, M.; Gard, A. L.; Xiang, F.; Wang, J.; Davoodian, N.; Lenz,
of bursicon in the animal kingdom. Gen. Comp. Endocrinol. 2006, P. H.; Malecha, S. R.; Christie, A. E.; Li, L. Combining in silico
27, 27. transcriptome mining and biological mass spectrometry for neuropep-
(44) Wilcockson, D. C.; Webster, S. G. Identification and develop- tide discovery in the Pacific white shrimp Litopenaeus vannamei. Peptides
mental expression of mRNAs encoding putative insect cuticle hardening 2010, 31 (1), 27–43.
hormone, bursicon in the green shore crab Carcinus maenas. Gen. Comp. (61) Christie, A. E.; Durkin, C. S.; Hartline, N.; Ohno, P.; Lenz, P. H.
Endocrinol. 2008, 156 (1), 113–125. Bioinformatic analyses of the publicly accessible crustacean expressed
(45) Sharp, J. H.; Wilcockson, D. C.; Webster, S. G. Identification sequence tags (ESTs) reveal numerous novel neuropeptide-encoding
and expression of mRNAs encoding bursicon in the plesiomorphic precursor proteins, including ones from members of several little studied
central nervous system of Homarus gammarus. Gen. Comp. Endocrinol. taxa. Gen. Comp. Endocrinol. 2010, 167 (1), 164–178.
2010, 169 (1), 65–74. (62) Phlippen, M. K.; Webster, S. G.; Chung, J. S.; Dircksen, H.
(46) Robertson, H. M.; Navik, J. A.; Walden, K. K.; Honegger, H. W. Ecdysis of decapod crustaceans is associated with a dramatic release of
The bursicon gene in mosquitoes: an unusual example of mRNA trans- crustacean cardioactive peptide into the haemolymph. J. Exp. Biol. 2000,
splicing. Genetics 2007, 176 (2), 1351–1353. 203 (Pt 3), 521–536.
(47) Z darek, J.; Nachman, R. J.; Denlinger, D. L. Parturition (63) Park, J. H.; Schroeder, A. J.; Helfrich-F€orster, C.; Jackson, F. R.;
hormone in the tsetse Glossina morsitans: activity in reproductive tissues Ewer, J. Targeted ablation of CCAP neuropeptide-containing neurons of
from other species and response of tsetse to identified neuropeptides and Drosophila causes specific defects in execution and circadian timing of
other neuroactive compounds. J. Insect Physiol. 2000, 46 (3), 213–219. ecdysis behavior. Development 2003, 130 (12), 2645–2656.
(48) Roller, L.; Yamanaka, N.; Watanabe, K.; Daubnerova, I.; Z it
nan, (64) Arakane, Y.; Li, B.; Muthukrishnan, S.; Beeman, R. W.; Kramer,
D.; Kataoka, H.; Tanaka, Y. The unique evolution of neuropeptide genes K. J.; Park, Y. Functional analysis of four neuropeptides, EH, ETH,
in the silkworm Bombyx mori. Insect Biochem. Mol. Biol. 2008, 38 (12), CCAP and bursicon, and their receptors in adult ecdysis behavior of the
1147–1157. red flour beetle, Tribolium castaneum. Mech. Dev. 2008, 125 (1112),
(49) Hansen, K. K.; Hauser, F.; Williamson, M.; Weber, S. B.; 984–995.
Grimmelikhuijzen, C. J. P. The Drosophila genes CG14593 and (65) Veenstra, J. A. Neurohormones and neuropeptides encoded by
CG30106 code for G-protein-coupled receptors specifically activated the genome of Lottia gigantea, with reference to other mollusks and
by the neuropeptides CCHamide-1 and CCHamide-2. Biochem. Biophys. insects. Gen. Comp. Endocrinol. 2010, 167 (1), 86–103.
Res. Commun. 2011, 404 (1), 184–189. (66) Chung, J. S.; Wilcockson, D. C.; Zmora, N.; Zohar, Y.;
(50) Predel, R.; Neupert, S.; Russell, W. K.; Scheibner, O.; Nachman, Dircksen, H.; Webster, S. G. Identification and developmental expres-
R. J. Corazonin in insects. Peptides 2007, 28 (1), 3–10. sion of mRNAs encoding crustacean cardioactive peptide (CCAP) in
(51) Tawfik, A. I.; Tanaka, S.; De Loof, A.; Schoofs, L.; Baggerman, decapod crustaceans. J. Exp. Biol. 2006, 209 (19), 3862–3872.
G.; Waelkens, E.; Derua, R.; Milner, Y.; Yerushalmi, Y.; Pener, M. P. (67) Loi, P. K.; Emmal, S. A.; Park, Y.; Tublitz, N. J. Identification,
Identification of the gregarization-associated dark-pigmentotropin in sequence and expression of a crustacean cardioactive peptide (CCAP) gene
locusts through an albino mutant. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 in the moth Manduca sexta. J. Exp. Biol. 2001, 204 (16), 2803–2816.
(12), 7083–7087. (68) G€ade, G. Regulation of intermediary metabolism and water
(52) Hoste, B.; Simpson, S. J.; Tanaka, S.; De Loof, A.; Breuer, M. A balance of insects by neuropeptides. Annu. Rev. Entomol. 2004,
comparison of phase-related shifts in behavior and morphometrics of an 49, 93–113.
albino strain, deficient in [His(7)]-corazonin, and a normally colored (69) Christie, A. E.; Stevens, J. S.; Bowers, M. R.; Chapline, M. C.;
Locusta migratoria strain. J. Insect Physiol. 2002, 48 (8), 791–801. Jensen, D. A.; Schegg, K. M.; Goldwaser, J.; Kwiatkowski, M. A.;
(53) Hoste, B.; Simpson, S. J.; Tanaka, S.; Zhu, D.; De Loof, A.; Pleasant, T. K., Jr.; Shoenfeld, L.; Tempest, L. K.; Williams, C. R.;
Breuer, M. Effects of [His(7)]-corazonin on the phase state of isolated- Wiwatpanit, T.; Smith, C. M.; Beale, K. M.; Towle, D. W.; Schooley,
reared (solitarious) desert locusts, Schistocerca gregaria. J. Insect Physiol. D. A.; Dickinson, P. S. Identification of a calcitonin-like diuretic
2002, 48 (10), 981–990. hormone that functions as an intrinsic modulator of the American
(54) Kim, Y. J.; Spalovska-Valachova, I.; Cho, K. H.; Z it
nanova, I.; lobster, Homarus americanus, cardiac neuromuscular system. J. Exp. Biol.
Park, Y.; Adams, M. E.; Z itnan, D. Corazonin receptor signaling in 2010, 213 (1), 118–127.
ecdysis initiation. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (17), (70) Roller, L.; Z it
nanova, I.; Dai, L.; Simo, L.; Park, Y.; Satake, H.;
6704–6709. Tanaka, Y.; Adams, M. E.; Z it
nan, D. Ecdysis triggering hormone
(55) Veenstra, J. A. Does corazonin signal nutritional stress in signaling in arthropods. Peptides 2010, 31 (3), 429–441.
insects? Insect Biochem. Mol. Biol. 2009, 39 (11), 755–762. (71) Predel, R.; Neupert, S.; Wicher, D.; Gundel, M.; Roth, S.; Derst,
(56) Boerjan, B.; Verleyen, P.; Huybrechts, J.; Schoofs, L.; De Loof, C. Unique accumulation of neuropeptides in an insect: FMRFamide-
A. In search for a common denominator for the diverse functions of related peptides in the cockroach, Periplaneta americana. Eur. J. Neurosci.
arthropod corazonin: a role in the physiology of stress? Gen. Comp. 2004, 20 (6), 1499–1513.
Endocrinol. 2010, 166 (2), 222–233. (72) Husson, S. J.; Mertens, I.; Janssen, T.; Lindemans, M.; Schoofs,
(57) Li, B.; Predel, R.; Neupert, S.; Hauser, F.; Tanaka, Y.; Cazzamali, L. Neuropeptidergic signaling in the nematode Caenorhabditis elegans.
G.; Williamson, M.; Arakane, Y.; Verleyen, P.; Schoofs, L.; Schachtner, Prog. Neurobiol. 2007, 82 (1), 33–55.
J.; Grimmelikhuijzen, C. J.; Park, Y. Genomics, transcriptomics, (73) Yamanaka, N.; Z it
nan, D.; Kim, Y. J.; Adams, M. E.; Hua, Y. J.;
and peptidomics of neuropeptides and protein hormones in the Suzuki, Y.; Suzuki, M.; Suzuki, A.; Satake, H.; Mizoguchi, A.; Asaoka, K.;
red flour beetle Tribolium castaneum. Genome Res. 2008, 18 (1), Tanaka, Y.; Kataoka, H. Regulation of insect steroid hormone biosynth-
113–122. esis by innervating peptidergic neurons. Proc. Natl. Acad. Sci. U.S.A.
(58) Huybrechts, J.; Bonhomme, J.; Minoli, S.; Prunier-Leterme, 2006, 103 (23), 8622–8627.
N.; Dombrovsky, A.; Abdel-Latief, M.; Robichon, A.; Veenstra, J. A.; (74) Nakabayashi, K.; Matsumi, H.; Bhalla, A.; Bae, J.; Mosselman,
Tagu, D. Neuropeptide and neurohormone precursors in the pea S.; Hsu, S. Y.; Hsueh, A. J. W. Thyrostimulin, a heterodimer of two new
aphid, Acyrthosiphon pisum. Insect Mol. Biol. 2010, 19 (Suppl 2), human glycoprotein hormone subunits, activates the thyroid-stimulating
87–95. hormone receptor. J. Clin. Invest. 2002, 109 (11), 1445–1452.
(59) Ma, M.; Bors, E. K.; Dickinson, E. S.; Kwiatkowski, M. A.; (75) Hsu, S. Y.; Nakabayashi, K.; Bhalla, A. Evolution of glycoprotein
Sousa, G. L.; Henry, R. P.; Smith, C. M.; Towle, D. W.; Christie, A. E.; Li, hormone subunit genes in bilateral metazoa: identification of two novel

human glycoprotein hormone subunit family genes, GPA2 and GPB5. (94) Egerod, K.; Reynisson, E.; Hauser, F.; Cazzamali, G.; Williamson,
Mol. Endocrinol. 2002, 16 (7), 1538–1551. M.; Grimmelikhuijzen, C. J. P. Molecular cloning and functional
(76) Hoyle, C. H. V. Neuropeptide families: evolutionary perspec- expression of the first two specific insect myosuppressin receptors. Proc.
tives. Regul. Pept. 1998, 73 (1), 1–33. Natl. Acad. Sci. U.S.A. 2003, 100 (17), 9808–9813.
(77) Stafflinger, E.; Hansen, K. K.; Hauser, F.; Schneider, M.; (95) Hauser, F.; Cazzamali, G.; Williamson, M.; Park, Y.; Li, B.;
Cazzamali, G.; Williamson, M.; Grimmelikhuijzen, C. J. P. Cloning Tanaka, Y.; Predel, R.; Neupert, S.; Schachtner, J.; Verleyen, P.;
and identification of an oxytocin/vasopressin-like receptor and its Grimmelikhuijzen, C. J. A genome-wide inventory of neurohormone
ligand from insects. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (9), GPCRs in the red flour beetle Tribolium castaneum. Front. Neuroendo-
3262–3267. crinol. 2008, 29 (1), 142–165.
(78) Proux, J. P.; Miller, C. A.; Li, J. P.; Carney, R. L.; Girardie, A.; (96) Holman, G. M.; Cook, B. J.; Nachman, R. J. Isolation, primary
Delaage, M.; Schooley, D. A. Identification of an arginine vasopressin- structure and synthesis of leucomyosuppressin, an insect neuropeptide
like diuretic hormone from Locusta migratoria. Biochem. Biophys. Res. that inhibits spontaneous contractions of the cockroach hindgut. Comp.
Commun. 1987, 149 (1), 180–186. Biochem. Physiol. C 1986, 85, 329–333.
(79) Aikins, M. J.; Schooley, D. A.; Begum, K.; Detheux, M.; Beeman, (97) Bendena, W. G.; Donly, B. C.; Fuse, M.; Lee, E.; Lange,
R. W.; Park, Y. Vasopressin-like peptide and its receptor function in an A. B.; Orchard, I.; Tobe, S. S. Molecular characterization of the
indirect diuretic signaling pathway in the red flour beetle. Insect Biochem. inhibitory myotropic peptide leucomyosuppressin. Peptides 1997, 18 (1),
Mol. Biol. 2008, 38 (7), 740–748. 157–163.
(80) Hauser, F.; Neupert, S.; Williamson, M.; Predel, R.; Tanaka, Y.; (98) Nichols, R.; Bendena, W. G.; Tobe, S. S. Myotropic peptides in
Grimmelikhuijzen, C. J. P. Genomics and peptidomics of neuropeptides Drosophila melanogaster and the genes that encode them. J. Neurogenet.
and protein hormones present in the parasitic wasp Nasonia vitripennis. 2002, 16 (1), 1–28.
J. Proteome Res. 2010, 9 (10), 5296–5310. (99) Stemmler, E. A.; Cashman, C. R.; Messinger, D. I.; Gardner,
(81) Wu, Q.; Brown, M. R. Signaling and function of insulin-like N. P.; Dickinson, P. S.; Christie, A. E. High-mass-resolution direct-tissue
peptides in insects. Annu. Rev. Entomol. 2006, 51, 1–24. MALDI-FTMS reveals broad conservation of three neuropeptides
(82) Nagasawa, H.; Kataoka, H.; Isogai, A.; Tamura, S.; Suzuki, A.; (APSGFLGMRamide, GYRKPPFNGSIFamide and pQDLDHVFLRF-
Mizoguchi, A.; Fujiwara, Y.; Takahashi, S. Y.; Ishizaki, H. Amino acid amide) across members of seven decapod crustaean infraorders. Peptides
sequence of a prothoracicotropic hormone of the silkworm Bombyx 2007, 28 (11), 2104–2115.
mori. Proc. Natl. Acad. Sci. U.S.A. 1986, 83 (16), 5840–5843. (100) Stevens, J. S.; Cashman, C. R.; Smith, C. M.; Beale, K. M.;
(83) Martin, G.; Sorokine, O.; Moniatte, M.; Vandorsselaer, A. The Towle, D. W.; Christie, A. E.; Dickinson, P. S. The peptide hormone
androgenic hormone of the crustacean isopod Armadillidium vulgare. pQDLDHVFLRFamide (crustacean myosuppressin) modulates the
Ann. N.Y. Acad. Sci. 1998, 839, 111–117. Homarus americanus cardiac neuromuscular system at multiple sites.
(84) Ohira, T.; Hasegawa, Y.; Tominaga, S.; Okuno, A.; J. Exp. Biol. 2009, 212 (Pt 24), 3961–3976.
Nagasawa, H. Molecular cloning and expression analysis of cDNAs (101) Badisco, L.; Claeys, I.; Van Loy, T.; Van Hiel, M.; Franssens,
encoding androgenic gland hormone precursors from two Porcel- V.; Simonet, G.; Vanden Broeck, J. Neuroparsins, a family of conserved
lionidae species, Porcellio scaber and P. dilatatus. Zool. Sci. 2003, 20 arthropod neuropeptides. Gen. Comp. Endocrinol. 2007, 153 (13),
(1), 75–81. 64–71.
(85) Manor, R.; Weil, S.; Oren, S.; Glazer, L.; Aflalo, E. D.; Ventura, (102) Badisco, L.; Claeys, I.; Van Hiel, M.; Clynen, E.; Huybrechts,
T.; Chalifa-Caspi, V.; Lapidot, M.; Sagi, A. Insulin and gender: an J.; Vandersmissen, T.; Van Soest, S.; Vanden Bosch, L.; Simonet, G.;
insulin-like gene expressed exclusively in the androgenic gland of the Vanden Broeck, J. Purification and characterization of an insulin-related
male crayfish. Gen. Comp. Endocrinol. 2007, 150 (2), 326–336. peptide in the desert locust, Schistocerca gregaria: immunolocalization,
(86) Ventura, T.; Manor, R.; Aflalo, E. D.; Weil, S.; Raviv, S.; Glazer, cDNA cloning, transcript profiling and interaction with neuroparsin.
L.; Sagi, A. Temporal silencing of an androgenic gland-specific insulin- J. Mol. Endocrinol. 2008, 40 (3), 137–150.
like gene affecting phenotypical gender differences and spermatogenesis. (103) Veenstra, J. A. What the loss of the hormone neuroparsin in
Endocrinology 2009, 150 (3), 1278–1286. the melanogaster subgroup of Drosophila can tell us about its function.
(87) Humbel, R. E. Insulin-like growth factors I and II. Eur. J. Insect Biochem. Mol. Biol. 2010, 40 (4), 354–361.
Biochem. 1990, 190 (3), 445–462. (104) Cerda-Reverter, J. M.; Larhammar, D. Neuropeptide Y family
(88) Chan, S. J.; Steiner, D. F. Insulin through the ages: phylogeny of of peptides: structure, anatomical expression, function, and molecular
a growth promoting metabolic hormone. Am. Zool. 2000, 40, 213–222. evolution. Biochem. Cell. Biol. 2000, 78 (3), 371–392.
(89) Clynen, E.; Huybrechts, J.; Baggerman, G.; Van Doorn, J.; Van (105) de Jong-Brink, M.; ter Maat, A.; Tensen, C. P. NPY in
Der Horst, D.; De Loof, A.; Schoofs, L. Identification of a glycogenolysis- invertebrates: molecular answers to altered functions during evolution.
inhibiting peptide from the corpora cardiaca of locusts. Endocrinology Peptides 2001, 22 (3), 309–315.
2003, 144 (8), 3441–3448. (106) Nuss, A. B.; Forschler, B. T.; Crim, J. W.; TeBrugge, V.; Pohl,
(90) Gr€onke, S.; Clarke, D. F.; Broughton, S.; Andrews, T. D.; J.; Brown, M. R. Molecular characterization of neuropeptide F from the
Partridge, L. Molecular evolution and functional characterization of eastern subterranean termite Reticulitermes flavipes (Kollar) (Isoptera:
Drosophila insulin-like peptides. PLoS Genet. 2010, 6 (2), e1000857. Rhinotermitidae). Peptides 2010, 31 (3), 419–428.
(91) Zhang, Q.; Keller, R.; Dircksen, H. Crustacean hyperglycaemic (107) N€assel, D. R.; Wegener, C. A comparative review of short and
hormone in the nervous system of the primitive crustacean species long neuropeptide F signaling in invertebrates: Any similarities to
Daphnia magna and Artemia salina (Crustacea: Branchiopoda). Cell vertebrate neuropeptide Y signaling? Peptides 2011, 32 (6), 1335–1355.
Tissue Res. 1997, 287 (3), 565–576. (108) Christie, A. E.; Cashman, C. R.; Brennan, H. R.; Ma, M.;
(92) Dircksen, H.; B€ocking, D.; Heyn, U.; Mandel, C.; Chung, J. S.; Sousa, G. L.; Li, L.; Stemmler, E. A.; Dickinson, P. S. Identification of
Baggerman, G.; Verhaert, P.; Daufeldt, S.; Pl€osch, T.; Jaros, P. P.; putative crustacean neuropeptides using in silico analyses of publicly
Waelkens, E.; Keller, R.; Webster, S. G. Crustacean hyperglycaemic accessible expressed sequence tags. Gen. Comp. Endocrinol. 2008, 156
hormone (CHH)-like peptides and CHH-precursor-related peptides (2), 246–264.
from pericardial organ neurosecretory cells in the shore crab, Carcinus (109) Clynen, E.; Husson, S. J.; Schoofs, L. Identification of new
maenas, are putatively spliced and modified products of multiple genes. members of the (short) neuropeptide F family in locusts and Caenor-
Biochem. J. 2001, 356, 159–170. habditis elegans. Ann. N.Y. Acad. Sci. 2009, 1163, 60–74.
(93) Dircksen, H. Insect ion transport peptides are derived from (110) Pascual, N.; Castresana, J.; Valero, M. L.; Andreu, D.; Belles, X.
alternatively spliced genes and differentially expressed in the central and Orcokinins in insects and other invertebrates. Insect Biochem. Mol. Biol.
peripheral nervous system. J. Exp. Biol. 2009, 212 (3), 401–412. 2004, 34 (11), 1141–1146.

(111) Hofer, S.; Dircksen, H.; Tollb€ack, P.; Homberg, U. Novel (128) Stemmler, E. A.; Bruns, E. A.; Gardner, N. P.; Dickinson, P. S.;
insect orcokinins: characterization and neuronal distribution in the Christie, A. E. Mass spectrometric identification of pEGFYSQRYamide: a
brains of selected dicondylian insects. J. Comp. Neurol. 2005, 490 (1), crustacean peptide hormone possessing a vertebrate neuropeptide Y
57–71. (NPY)-like carboxy-terminus. Gen. Comp. Endocrinol. 2007, 152 (1), 1–7.
(112) Yasuda-Kamatani, Y.; Yasuda, A. Identification of orcokinin (129) Janssen, I.; Schoofs, L.; Spittaels, K.; Neven, H.; Vanden
gene-related peptides in the brain of the crayfish Procambarus clarkii by Broeck, J.; Devreese, B.; Van Beeumen, J.; Shabanowitz, J.; Hunt,
the combination of MALDI-TOF and on-line capillary HPLC/Q-Tof D. F.; De Loof, A. Isolation of NEB-LFamide, a novel myotropic
mass spectrometries and molecular cloning. Gen. Comp. Endocrinol. neuropeptide from the grey fleshfly. Mol. Cell. Endocrinol. 1996, 117
2000, 118 (1), 161–172. (2), 157–165.
(113) Dickinson, P. S.; Stemmler, E. A.; Barton, E. E.; Cashman, (130) Verleyen, P.; Huybrechts, J.; Schoofs, L. SIFamide illustrates
C. R.; Gardner, N. P.; Rus, S.; Brennan, H. R.; McClintock, T. S.; the rapid evolution in arthropod neuropeptide research. Gen. Comp.
Christie, A. E. Molecular, mass spectral, and physiological analyses of Endocrinol. 2009, 162 (1), 27–35.
orcokinins and orcokinin precursor-related peptides in the lobster (131) Terhzaz, S.; Rosay, P.; Goodwin, S. F.; Veenstra, J. A. The
Homarus americanus and the crayfish Procambarus clarkii. Peptides neuropeptide SIFamide modulates sexual behavior in Drosophila. Bio-
2009, 30 (2), 297–317. chem. Biophys. Res. Commun. 2007, 352 (2), 305–310.
(114) Predel, R.; Wegener, C. Biology of the CAPA peptides in (132) Sithigorngul, P.; Pupuem, J.; Krungkasem, C.; Longyant, S.;
insects. Cell. Mol. Life Sci. 2006, 63 (21), 2477–2490. Chaivisuthangkura, P.; Sithigorngul, W.; Petsom, A. Seven novel
(115) Kean, L.; Cazenave, W.; Costes, L.; Broderick, K. E.; Graham, FMRFamide-like neuropeptide sequences from the eyestalk of the giant
S.; Pollock, V. P.; Davies, S. A.; Veenstra, J. A.; Dow, J. A. T. Two tiger prawn Penaeus monodon. Comp. Biochem. Physiol., B: Biochem. Mol.
nitridergic peptides are encoded by the gene capability in Drosophila Biol. 2002, 131 (3), 325–337.
melanogaster. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002, 282 (5), (133) Yasuda, A.; Yasuda-Kamatani, Y.; Nozaki, M.; Nakajima, T.
R1297–1307. Identification of GYRKPPFNGSIFamide (crustacean-SIFamide) in the
(116) Choi, M.-Y.; Vander Meer, R. K.; Valles, S. M. Molecular crayfish Procambarus clarkii by topological mass spectrometry analysis.
diversity of PBAN family peptides from fire ants. Arch. Insect Biochem. Gen. Comp. Endocrinol. 2004, 135 (3), 391–400.
Physiol. 2010, 74 (2), 67–80. (134) Dickinson, P. S.; Stemmler, E. A.; Cashman, C. R.; Brennan,
(117) Torfs, P.; Nieto, J.; Cerstiaens, A.; Boon, D.; Baggerman, G.; H. R.; Dennison, B.; Huber, K. E.; Peguero, B.; Rabacal, W.; Goiney,
Poulos, C.; Waelkens, E.; Derua, R.; Calderon, J.; De Loof, A.; Schoofs, L. C. C.; Smith, C. M.; Towle, D. W.; Christie, A. E. SIFamide peptides in
Pyrokinin neuropeptides in a crustacean. Isolation and identification in the clawed lobsters and freshwater crayfish (Crustacea, Decapoda,
white shrimp Penaeus vannamei. Eur. J. Biochem. 2001, 268 (1), 149–154. Astacidea): a combined molecular, mass spectrometric and electrophy-
(118) Ma, M.; Chen, R.; Sousa, G. L.; Bors, E. K.; Kwiatkowski, siological investigation. Gen. Comp. Endocrinol. 2008, 156 (2), 347–360.
M. A.; Goiney, C. C.; Goy, M. F.; Christie, A. E.; Li, L. Mass spectral (135) Spittaels, K.; Verhaert, P.; Shaw, C.; Johnston, R. N.; Devreese,
characterization of peptide transmitters/hormones in the nervous B.; Van Beeumen, J.; De Loof, A. Insect neuropeptide F (NPF)-related
system and neuroendocrine organs of the American lobster Homarus peptides: isolation from Colorado potato beetle (Leptinotarsa decemlineata)
americanus. Gen. Comp. Endocrinol. 2008, 156 (2), 395–409. brain. Insect Biochem. Mol. Biol. 1996, 26 (4), 375–382.
(119) Ma, M.; Wang, J.; Chen, R.; Li, L. Expanding the crustacean (136) Sithigorngul, P.; Saraithongkum, W.; Jaideechoey, S.; Longyant,
neuropeptidome using a multifaceted mass spectrometric approach. S.; Sithigorngul, W. Novel FMRFamide-like neuropeptides from the eyestalk
J. Proteome Res. 2009, 8 (5), 2426–2437. of the giant freshwater prawn Macrobrachium rosenbergii. Comp. Biochem.
(120) Strauss, J.; Zhang, Q.; Verleyen, P.; Huybrechts, J.; Neupert, Physiol., B: Biochem. Mol. Biol. 1998, 120, 587–595.
S.; Predel, R.; Pauwels, K.; Dircksen, H. Pigment-dispersing hormone in (137) Dickinson, P. S.; Stevens, J. S.; Rus, S.; Brennan, H. R.; Goiney,
Daphnia brain and visual ganglia interneurons, one type homologous to C. C.; Smith, C. M.; Li, L.; Towle, D. W.; Christie, A. E. Identification
insect clock neurons displaying circadian rhythmicity. Cell. Molec. Life and cardiotropic actions of sulfakinin peptides in the American lobster
Sci. 2011, DOI: 10.1007/s00018-011-0636-3 . Homarus americanus. J. Exp. Biol. 2007, 210 (13), 2278–2289.
(121) Rao, K. R.; Riehm, J. P. Pigment-dispersing hormones: a novel (138) Christie, A. E. Neuropeptide discovery in Ixodoidea: an in
family of neuropeptides from arthropods. Peptides 1988, 1, 153–159. silico investigation using publicly accessible expressed sequence tags.
(122) Rao, K. R. Crustacean pigmentary-effector hormones: Chem- Gen. Comp. Endocrinol. 2008, 157 (2), 174–185.
istry and functions of RPCH, PDH, and related peptides. Am. Zool. (139) Nichols, R.; Manoogian, B.; Walling, E.; Mispelon, M. Plasti-
2001, 41 (3), 364–379. city in the effects of sulfated and nonsulfated sulfakinin on heart
(123) Bulau, P.; Meisen, I.; Schmitz, T.; Keller, R.; Peter-Katalinic, J. contractions. Front. Biosci. 2009, 14, 4035–4043.
Identification of neuropeptides from the sinus gland of the crayfish (140) Van Loy, T.; Vandersmissen, H. P.; Poels, J.; Van Hiel, M. B.;
Orconectes limosus using nanoscale on-line liquid chromatography tan- Verlinden, H.; Vanden Broeck, J. Tachykinin-related peptides and their
dem mass spectrometry. Mol. Cell. Proteomics 2004, 3 (6), 558–564. receptors in invertebrates: a current view. Peptides 2010, 31 (3), 520–524.
(124) Isaac, R. E.; Taylor, C. A.; Hamasaka, Y.; N€assel, D. R.; Shirras, (141) Christie, A. E.; Lundquist, C. T.; N€assel, D. R.; Nusbaum,
A. D. Proctolin in the post-genomic era: new insights and challenges. M. P. Two novel tachykinin-related peptides from the nervous system of
Invert. Neurosci. 2004, 5 (2), 51–64. the crab Cancer borealis. J. Exp. Biol. 1997, 200 (17), 2279–2294.
(125) Taylor, C. A.; Winther, A. M.; Siviter, R. J.; Shirras, A. D.; (142) Nieto, J.; Veelaert, D.; Derua, R.; Waelkens, E.; Cerstiaens, A.;
Isaac, R. E.; N€assel, D. R. Identification of a proctolin preprohormone Coast, G.; Devreese, B.; Van Beeumen, J.; Calderon, J.; De Loof, A.;
gene (Proct) of Drosophila melanogaster: expression and predicted Schoofs, L. , Identification of one tachykinin- and two kinin-related
prohormone processing. J. Neurobiol. 2004, 58 (3), 379–391. peptides in the brain of the white shrimp, Penaeus vannamei. Biochem.
(126) Spittaels, K.; Vankeerberghen, A.; Torrekens, S.; Devreese, B.; Biophys. Res. Commun. 1998, 248 (2), 406–411.
Grauwels, L.; Van Leuven, F.; Hunt, D.; Shabanowitz, J.; Schoofs, L.; (143) Christie, A. E.; Kutz-Naber, K. K.; Stemmler, E. A.; Klein, A.;
Van Beeumen, J.; De Loof, A. Isolation of Ala1-proctolin, the first natural Messinger, D. I.; Goiney, C. C.; Conterato, A. J.; Bruns, E. A.; Hsu, Y. W.;
analogue of proctolin, from the brain of the Colorado potato beetle. Mol. Li, L.; Dickinson, P. S. Midgut epithelial endocrine cells are a rich source
Cell. Endocrinol. 1995, 110 (12), 119–124. of the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-
(127) Fu, Q.; Kutz, K. K.; Schmidt, J. J.; Hsu, Y. W.; Messinger, D. I.; related peptide Ia) and GYRKPPFNGSIFamide (Gly1-SIFamide) in the
Cain, S. D.; de la Iglesia, H. O.; Christie, A. E.; Li, L. Hormone crabs Cancer borealis, Cancer magister and Cancer productus. J. Exp. Biol.
complement of the Cancer productus sinus gland and pericardial organ: 2007, 210 (Pt 4), 699–714.
an anatomical and mass spectrometric investigation. J. Comp. Neurol. (144) N€assel, D. R. Tachykinin-related peptides in invertebrates: a
2005, 493 (4), 607–626. review. Peptides 1999, 20 (1), 141–158.

(145) Taylor, P. A., 3rd; Bhatt, T. R.; Horodyski, F. M. Molecular

characterization and expression analysis of Manduca sexta allatotropin.
Eur. J. Biochem. 1996, 239 (3), 588–596.
(146) Horodyski, F. M.; Bhatt, S. R.; Lee, K. Y. Alternative splicing of
transcripts expressed by the Manduca sexta allatotropin (Mas-AT) gene
is regulated in a tissue-specific manner. Peptides 2001, 22 (2), 263–269.
(147) Montagne, N.; Desdevises, Y.; Soyez, D.; Toullec, J. Y.
Molecular evolution of the crustacean hyperglycemic hormone family
in ecdysozoans. BMC Evol. Biol. 2010, 10, 62.
(148) Li, W.; Tucker, A. E.; Sung, W.; Thomas, W. K.; Lynch, M.
Extensive, recent intron gains in Daphnia populations. Science 2009, 326
(5957), 1260–1262.
(149) Telford, M. J.; Bourlat, S. J.; Economou, A.; Papillon, D.; Rota-
Stabelli, O. The evolution of the Ecdysozoa. Philos. Trans. R. Soc. Lond.,
B: Biol. Sci. 2008, 363 (1496), 1529–1537.
(150) Regier, J. C.; Shultz, J. W.; Zwick, A.; Hussey, A.; Ball, B.;
Wetzer, R.; Martin, J. W.; Cunningham, C. W. Arthropod relationships
revealed by phylogenomic analysis of nuclear protein-coding sequences.
Nature 2010, 463 (7284), 1079–1083.
(151) Kato, Y.; Shiga, Y.; Kobayashi, K.; Tokishita, S. I.; Yamagata,
H.; Iguchi, T.; Watanabe, H. Development of an RNA interference
method in the cladoceran crustacean Daphnia magna. Dev. Genes Evol.
2011, 220 (1112), 337–345.
(152) Predel, R. Peptidergic neurohemal system of an insect: mass
spectrometric morphology. J. Comp. Neurol. 2001,
436 (3), 363–375.
(153) Williamson, M.; Lenz, C.; Winther, A. M.; N€assel, D. R.;
Grimmelikhuijzen, C. J. Molecular cloning, genomic organization, and
expression of a B-type (cricket-type) allatostatin preprohormone from
Drosophila melanogaster. Biochem. Biophys. Res. Commun. 2001, 281 (2),
544–550.
(154) Neupert, S.; Schattschneider, S.; Predel, R. Allatotropin-
related peptide in cockroaches: identification via mass spectrometric
analysis of single identified neurons. Peptides 2009, 30 (3), 489–494.
(155) 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. Bio-
chemistry 2009, 48 (38), 9047–9060.
(156) Veenstra, J. A.; Lambrou, G. Isolation of a novel RFamide
peptide from the midgut of the American cockroach, Periplaneta
americana. Biochem. Biophys. Res. Commun. 1995, 213 (2), 519–524.
(157) Gaus, G.; Doble, K. E.; Price, D. A.; Greenberg, M. J.; Lee,
T. D.; Battelle, B. A. The sequences of five neuropeptides isolated from
Limulus using antisera to FMRFamide. Biol. Bull. 1993, 184 (3),
322–329.
(158) Huybrechts, J.; Nusbaum, M. P.; Vanden Bosch, L.; Baggerman,
G.; De Loof, A.; Schoofs, L. Neuropeptidomic analysis of the brain and
thoracic ganglion from the Jonah crab, Cancer borealis. Biochem. Biophys.
Res. Commun. 2003, 308 (3), 535–544.

TMP BD5 E

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

TMP BD5 E

Uploaded by

Copyright:

Available Formats

ARTICLE

Genomics, Transcriptomics, and Peptidomics of Daphnia pulex

Adipokinetic hormone pQVNFSTSWa 950.44 + +/nd

4480 dx.doi.org/10.1021/pr200284e |J. Proteome Res. 2011, 10, 4478–4504

predicted neuropeptides from 45 precursors (Table 1; Suppl.

middle part of the peptides to an AT-like L. migratoria abdominal

Information), which, however, conﬁrms the expression of the CCHamide

similarities were found for the body louse Pediculus humanus,

SIFamide always occurs directly after the SP followed by a so-

4499 dx.doi.org/10.1021/pr200284e |J. Proteome Res. 2011, 10, 4478–4504

4500 dx.doi.org/10.1021/pr200284e |J. Proteome Res. 2011, 10, 4478–4504

4501 dx.doi.org/10.1021/pr200284e |J. Proteome Res. 2011, 10, 4478–4504

4502 dx.doi.org/10.1021/pr200284e |J. Proteome Res. 2011, 10, 4478–4504

4503 dx.doi.org/10.1021/pr200284e |J. Proteome Res. 2011, 10, 4478–4504

(145) Taylor, P. A., 3rd; Bhatt, T. R.; Horodyski, F. M. Molecular

4504 dx.doi.org/10.1021/pr200284e |J. Proteome Res. 2011, 10, 4478–4504

You might also like