mass spectrometric characterization of the neuropeptidome of the ghost crab ocypode ceratophthalma...

General and Comparative Endocrinology 184 (2013) 22–34

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General and Comparative Endocrinology

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Mass spectrometric characterization of the neuropeptidome of the ghost crabOcypode ceratophthalma (Brachyura, Ocypodidae)

Limei Hui a, Brandon T. D’Andrea b, Chenxi Jia c, Zhidan Liang c, Andrew E. Christie b,⇑, Lingjun Li a,c,⇑a Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706-1396, USAb Békésy Laboratory of Neurobiology, Pacific Biosciences Research Center, University of Hawaii at Manoa, 1993 East–West Road, Honolulu, HI 96822, USAc School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705-2222, USA

a r t i c l e i n f o

Article history:Received 17 August 2012Revised 17 December 2012Accepted 18 December 2012Available online 5 January 2013

Keywords:MALDI-TOF/TOF mass spectrometryNanoLC-ESI-Q-TOF tandem massspectrometryNeuropeptideNeurohormoneCrustaceaDe novo sequencing

0016-6480/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ygcen.2012.12.008

⇑ Corresponding authors: Fax: +1 608 262 5345 (Christie)

E-mail addresses: [email protected] (A.E.c.edu (L. Li).

a b s t r a c t

The horn-eyed ghost crab Ocypode ceratophthalma is a terrestrial brachyuran native to the Indo-Pacificregion, including the islands of Hawaii. Here, multiple mass spectrometric platforms, including matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) and nanoflow liquid chromatography coupled with electrospray ionization quadrupoletime-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS), were used to characterize theneuropeptidome of this species. In total, 156 peptide paracrines/hormones, representing 15 peptide fam-ilies, were identified from the O. ceratophthalma supraesophageal ganglion (brain), eyestalk ganglia, peri-cardial organ and/or sinus gland, including 59 neuropeptides de novo sequenced here for the first time.Among the de novo sequenced peptides were isoforms of A-type allatostatin, B-type allatostatin, FMRFa-mide-like peptide (FLP), orcokinin, orcomyotropin and RYamide. Of particular note, were several novelFLPs including DVRAPALRLRFamide, an isoform of short neuropeptide F, and NRSNLRFamide, the orcoki-nins NFDEIDRSGYGFV and DFDEIDRSSFGFH, which exhibit novel Y for F and D for N substitutions at posi-tions 10 and 1, respectively, and FDAYTTGFGHS, a member of the orcomyotropin family exhibiting anovel Y for F substitution at position 4. Taken collectively, the set of peptides described here representsthe largest number of neuropeptides thus far characterized via mass spectrometry from any single crus-tacean, and provides a framework for future investigations of the physiological roles played by these mol-ecules in this species.

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1. Introduction

A large number of molecules are used by nervous systems forchemical communication. These substances can be divided intoseveral broad classes, the largest and most diverse of which isthe peptides. While many organisms have contributed to ourknowledge of peptidergic signaling, work done on crustaceanshas provided many important insights for over half a century[6,8,9]. For example, peptidergic neurosecretion was first formallydemonstrated using a crustacean model [1,45], and the first inver-tebrate neuropeptide to be isolated and fully characterized wasalso from a member of this arthropod subphylum [17]. More re-cently, the crustacean stomatogastric and cardiac neuromuscularsystems have been invaluable contributors to our understandingof the generation, maintenance, and modulation of rhythmicallyactive behaviors. Specifically, these systems have been instrumen-

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L. Li), +1 808 956 6984 (A.E.

Christie), [email protected]

tal in discerning how locally-released and circulating neuropep-tides allow for the production of an essentially infinite number ofdistinct behavioral outputs from a single, numerically simple,‘‘hard-wired’’, neural network [6,9,11,16,21,25,41–44,48,50,53]. Inaddition to their use as biomedical models, the diversity of specieswhich comprise the Crustacea makes this group of animals a con-venient model in which to assess the structural and functional evo-lution of peptides/peptide families [9]; there are approximately67,000 extant species in this arthropod subphylum and its mem-bers inhabit many highly diverse aquatic and terrestrial environ-ments [2].

Prior to the early-2000s, the common strategy for crustaceanpeptide identification was to isolate a single neuropeptide bio-chemically/chromatographically from a large pool of starting tis-sue, and then to determine its structure using a combination ofproteolytic cleavage and Edman analysis [6,9]. Recently, therehas been a shift in the focus of crustacean neuropeptide discoveryfrom the identification and structural determination of single pep-tides to the characterization of entire peptidomes, the full comple-ment of neuropeptides used by an animal [6,9]. Mass spectrometry,with its highly sensitive, accurate mass matching and de novo

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L. Hui et al. / General and Comparative Endocrinology 184 (2013) 22–34 23

sequencing capabilities, has been a major contributor to this shiftin focus for the field [6,9,34].

At present, members of the Decapoda are by far the most thor-oughly investigated crustaceans in terms of their neuropeptides[6,9]. This said, only a handful of species have been the subjectsof large-scale mass spectrometric analyses, i.e. the penaeid shrimpLitopenaeus vannamei [38], the astacidean lobster Homarus americ-anus [4,5,37], and the brachyuran crabs Cancer borealis [29,33,40],Cancer productus [18,19], Carcinus maenas [36] and Callinectes sapi-dus [26–28], all of which are marine. The horn-eyed ghost crab,Ocypode ceratophthalma, is distinct from the other decapods whosepeptidomes have been deduced in that it is primarily a terrestrialspecies, inhabiting the supralittoral zone of sandy beachesthroughout the Indo-Pacific region [22]. Here, we present a mul-ti-platform mass spectral characterization of the neuropeptidomeof O. ceratophthalma (derived from selected portions of the centralnervous system [supraesophageal and eyestalk ganglia], as well asits primary neuroendocrine organs [sinus gland and pericardial or-gan]), comparing it to those of the other decapods thus farsurveyed.

2. Materials and methods

2.1. Animals

Horn-eyed ghost crabs, O. ceratophthalma, were collected byhand from the supralittoral zones of Malaekahana Beach (Laie,Oahu, HI) and the beach at Maunalua Bay adjacent to Paiko La-goon/Kuli’ou’ou Beach Park (Honolulu, Oahu, HI); all animals weremaintained in moist sand at room temperature (�22 �C) until usedfor tissue collection (generally less than 24 h after collection).

2.2. Tissue collection

Prior to dissection, crabs were anesthetized by packing in ice forapproximately 15 min, after which the eyestalk ganglia (includingthe sinus gland [SG]), supraesophageal ganglia (brain), pericardialorgans (POs) and/or individual SGs were isolated by manual micro-dissection and immediately placed in ice-cold acidified methanol,i.e. 90% methanol (Fisher Scientific, Pittsburgh, PA; catalog No.:AC61009-0040 HPLC Grade)/9% glacial acetic acid (Fisher; catalogNo.: A38S-212)/1% water (prepared using a PURELAB Plus Ultra-pure water filtration system [ELGA LabWater LLC, Woodridge,IL]). For all experiments, pooled tissues were collected: eyestalkganglia, 30 in 500 lL of acidified methanol; brains, 30 in 500 lLof acidified methanol; SGs, 30 in 100 lL of acidified methanol;POs, 30 in 100 lL of acidified methanol. All pooled samples werestored at �80 �C until used for tissue extraction.

2.3. Tissue extraction

Pooled tissues were homogenized in cold acidified methanol(see Section 2.2) using a handheld ground glass homogenizer(Wheaten Science; Millville, NJ), after which the tissue homoge-nate was centrifuged at 16,000�g using an Eppendorf 5415D table-top centrifuge (Eppendorf AG, Hauppauge, NY). The resultingsupernatant was collected and dried using a Savant SC 110 Speed-Vac concentrator (Thermo Scientific, Asheville, NC) and then re-suspended in 50 lL of 0.1% formic acid (Fisher; catalog No.:AC14793-2500) in water for further separation and analysis.

2.4. Reversed phase-HPLC fractionation of tissue extracts

The extracts produced for each tissue were fractionated via highperformance liquid chromatography (HPLC). Specifically, the re-

suspended tissue supernatants described earlier (see Section 2.3)were vortexed and briefly centrifuged, after which they were sub-jected to separation using a Rainin Dynamax HPLC systemequipped with a UV-D II absorbance detector (Rainin InstrumentInc., Woburn, MA). The mobile phases used for HPLC fractionationwere de-ionized water containing 0.1% formic acid (Solution A) andacetonitrile (ACN; Fisher; catalog No.: AC610010040) containing0.1% formic acid (Solution B). Approximately 50 lL of extract wasinjected onto a Macrosphere C18 column (2.1 mm i.d. � 250 mmlength, 5 lm particle size; Alltech Assoc. Inc., Deerfield, IL). HPLCseparation consisted of a 120-min gradient of 5–95% Solution B,with fractions automatically collected every two minutes using aRainin Dynamax FC-4 fraction collector. Following separation, eachfraction was dried using a Savant SC 110 SpeedVac concentratorand then resuspended in 10 lL of 0.1% formic acid.

2.5. MALDI-TOF/TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was performedusing an Autoflex III TOF/TOF analyzer (Bruker Daltonics, Billerica,MA) equipped with a 200 Hz laser; this instrument was used foroff-line HPLC fraction screening. Off-line analysis of HPLC fractionswas performed by spotting 0.4 lL of an HPLC fraction (seeSection 2.4) on the MALDI sample plate and adding 0.4 lL of5 mg/mL a-cyano-4-hydroxy-cinnamic acid (CHCA); the resultingmixture was allowed to crystallize at room temperature (approxi-mately 20 �C). Two thousand laser shots were collected per samplespot at an accelerating voltage of 19 kV and a constant laser powerusing random shot selection. The acquired data covering m/z500–4000 were analyzed using FlexAnalysis software (Bruker Dal-tonics). The peptide identification was made by accurate massmatching against a home-built database consisting of publishedcrustacean neuropeptide sequences.

2.6. NanoLC-ESI-Q-TOF mass spectrometry

2.6.1. Q-TOF Micromass spectrometer systemFor some experiments, nanoflow liquid chromatography cou-

pled with electrospray ionization quadrupole time-of-flight tan-dem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS) wasperformed using a Waters nanoAcquity UPLC system coupled to aQ-TOF Micromass spectrometer (Waters Corporation, Milford,MA). Here, an in-house prepared, C18 reversed phase capillary col-umn (75 lm internal diameter � 100 mm length, 3 lm particlesize, 100 Å pore size) was used for the second dimension separa-tion of the HPLC described above. The mobile phases used for sep-aration were: 0.1% formic acid in deionized water (mobile phase A)and 0.1% formic acid in ACN (mobile phase B). An aliquot of 5.0 lLof a tissue extract or HPLC fraction was injected and loaded ontothe trap column (Zorbax 300SB-C18 Nano trapping column; Agi-lent Technologies, Santa Clara, CA) using mobile phase A at a flowrate of 10 lL/min for 10 min, after which the stream select modulewas switched to a position at which the trap column came in linewith the analytical capillary column, and a linear gradient of 5–45%mobile phase B over 90 min was initiated. The nanoflow ESI sourceconditions were set as follows: capillary voltage 3200 V, samplecone voltage 35 V, extraction cone voltage 1 V, source temperature100 �C; data dependent acquisition was employed for the MS sur-vey scan, selection of three precursor ions, and subsequent MS/MSof the selected parent ions. The MS scan range was from m/z 400 to1800, and the MS/MS scan was from m/z 50 to 1800.

2.6.2. Synapt G2 HDMS mass spectrometer systemIn addition to the system described in Section 2.6.1, nanoLC-ESI-

Q-TOF MS/MS was also performed using a Waters nanoAcquity

24 L. Hui et al. / General and Comparative Endocrinology 184 (2013) 22–34

UPLC system coupled to a Synapt G2 HDMS mass spectrometer(Waters). Here, chromatographic separations were performed ona Waters BEH 130 Å C18 reversed phase capillary column(150 mm � 75 lm, 1.7 lm). The mobile phases used were thesame as those described in Section 2.6.1. An aliquot of 2.0 lL ofan HPLC fraction was injected and loaded onto the Waters Nano-Ease trap column using 95% mobile phase A and 5% mobile phaseB at a flow rate of 10 lL/min for 3 min. Following this loading, alinear gradient from 5% to 45% mobile phase B over 75 min wasemployed for separation. Data dependent acquisition was em-ployed for the MS survey scan, the selection of three precursorions, and subsequent MS/MS of the selected parent ions. The MSscan range was from m/z 400 to 2000, and the MS/MS scan wasfrom m/z 50 to 2000.

3. Results

To determine the neuropeptidome of O. ceratophthalma, a strat-egy combining multiple mass spectral platforms was used. In thesections that follow, the identified peptides have been groupedinto families of related isoforms and these are presented belowin alphabetical order based on family name. Table 1 shows the se-quences of the identified peptides, as well as the mass spectralplatform(s) through which they were identified; Table 1 also pro-vides information on the tissues in which the peptides were de-tected. Fig. 1 shows examples of the MALDI-TOF/TOF MS spectraused for the identification of known peptides based on accuratemass matching of predicted vs. detected mass-to-charge ratios(m/z). This figure also highlights the power of HPLC for reducingthe chemical complexity of crude tissue extracts, thus allowingfor increased peptide identification in fractionated samples. Theremaining mass spectra presented here show examples of de novopeptide sequencing using the nanoLC-ESI-Q-TOF platform. In-cluded in several figures (e.g. Fig. 2) are MS/MS spectra generatedfor the same peptide using each of the two instruments used inour study: Q-TOF Micromass and Synapt G2 HDMS; these spectrahighlight the distinct, and complementary, information that eachof the nanoLC-ESI-Q-TOF systems provides for de novo sequenceassignment.

3.1. A-type allatostatin

Sixteen peptides possessing –YXFGLamide carboxyl (C)-termini(where X represents a variable residue), the hallmark of the A-typeallatostatin (A-AST) family [9], were identified from O. ceratophth-alma neural extracts (Table 1). Of these peptides, 12 were isoformsknown from other species, for example RGPYAFGLamide, FSGASPY-GLamide (Fig. 1B) and TRPYSFGLamide, all of which are known C.maenas isoforms [36]. The remaining four A-ASTs, SGHYIFGLamide(Fig. 1B), EPQYSFGLamide, KLPTGYQFGLamide (Fig. 1B), and YDF-EASSYSFGLamide, were identified here for the first time.

A-ASTs were identified in each of the tissues analyzed (Table 1).However, the PO was by far the richest source of members of thispeptide family, with nine of the 16 isoforms detected in this tissue(Table 1). In contrast, the SG had the fewest A-ASTs, with just twopeptides identified in this neuroendocrine organ (Table 1). Inter-estingly, only three of the 16 A-AST isoforms were identified frommultiple tissues (Table 1).

3.2. B-type allatostatin

Fifteen peptides possessing the C-terminal motif –WX6Wamide(where X6 represents six variable amino acids), the hallmark of theB-type allatostatin (B-AST) family [9], were identified in O. cerat-ophthalma (Table 1). Of these peptides, nine, DGSSNLRGAWamide,

TGWSSTSRAWamide, TGWSVFQGSWamide, TSWGKYQGSWamide,SDGWSSLRGSWamide, TQWSKFQGSWamide, DDWSQFQGSWa-mide, STNWSSCPTSAWamide and SPNDWAHFRGSWamide(Fig. 2) are novel B-AST isoforms, sequenced here for the first time.The remaining six B-ASTs are known crustacean isoforms, e.g.AWSNLGQAWamide, which was originally identified from C. mae-nas [36].

The PO was by far the richest source of B-ASTs, with all 14 iso-forms present in this tissue (Table 1). Seven B-ASTs were present inthe brain, with four sequenced from the eyestalk ganglia (Table 1).Interestingly, no B-type peptides were detected in the SG (Table 1).Eight of the 14 B-ASTs were present in multiple tissues (Table 1).

3.3. Crustacean cardioactive peptide

The peptide PFCNAFTGCamide, commonly referred to as crusta-cean cardioactive peptide or CCAP [9], was identified in both thebrain and PO of O. ceratophthalma (Table 1); this peptide has pre-viously been identified in a large number of decapod species (e.g.[10,19,26,27,33,37,38,40,52]).

3.4. Crustacean hyperglycemic hormone precursor-related peptide

Ten novel, apparently truncated, crustacean hyperglycemic hor-mone precursor-related peptides (CPRPs), LPSAHPLE (Fig. 3A),GPAESSGESAHPLE, RGPAESSGESAHPLE, SLRGPAESSGESALSE(Fig. 3B), LRGPAESSGESAHPLE, SLRGPAESSGESAHPLE, SLRGPAESDGESAHPLE (Fig. 3C), TSLRGPAESSGESAHPLE, LTSLRGPAESSGESAHPLE and LLTSLRGPAESSGESAHPLE (Fig. 3D), were sequencedfrom the eyestalk ganglia (5 of the 10 peptides) and/or SG (8 of the10 truncated CPRPs) of O. ceratophthalma (Table 1). Three of the 10peptides were sequenced from both tissues (Table 1).

Alignment of the 10 truncated CPRPs (Fig. 4) suggests that atleast four full-length peptides are produced in the O. ceratophth-alma eyestalk, with seven of the 10 peptides likely derived fromone full-length CPRP, and the remaining three truncations repre-senting portions of three other distinct CPRPs. This complementof four full-length CPRPs, if complete, would be identical to thatseen in the XO–SG systems of several other brachyurans, for exam-ple C. productus [18,24,57].

3.5. FMRFamide-like peptide

In crustaceans, a number of distinct subgroups of peptides formwhat is commonly referred to as the FMRFamide-like peptide (FLP)or RFamide superfamily [9]. The FLP subgroups include the myo-suppressin (–HVFLRFamide C-terminal consensus motif), neuro-peptide F (typically 36 amino acids in overall length andpossessing the C-terminal motif –GRPRFamide), short neuropep-tide F (sNPF; typically �10 amino acids long and possessing –RXRFamide C-termini, where X is a variable residue) and sulfakinin(possessing the C-terminal consensus motif –Y(SO3H)GHM/LRFa-mide) subfamilies, as well as a number of other N-terminally ex-tended –(F/Y)(L/V/I)RFamides [8,9]. As described in detail below,a total of 30 FLPs were sequenced from the neural extracts of O.ceratophthalma (Table 1), including eight novel members of thispeptide superfamily.

3.5.1. MyosuppressinTwo peptides possessing –HVFLRFamide C-termini,

pQDLDHVFLRFamide (Fig. 1A) and QDLDHVFLRFamide, were iden-tified from the brain, eyestalk ganglia and SG of O. ceratophthalma(Table 1). Both myosuppressins are known, broadly conservedcrustacean isoforms (e.g. [37,56]).

Table 1Ocypode ceratophthalma neuropeptides identified via mass spectrometry.

(continued on next page)


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3.5.2. Short neuropeptide FFour peptides exhibiting –RLRFamide C-termini were identified

from the eyestalk ganglia (all four isoforms), brain (two of the fourpeptides) and/or SG (one peptide) of O. ceratophthalma (Table 1).Three of the four sNPFs are known crustacean isoforms, e.g.SMPSLRLRFamide, a broadly conserved crustacean sNPF (e.g.[29,36–38,40,49]). The remaining sNPF, DVRAPALRLRFamide(Fig. 5A), is novel, being sequenced from the eyestalk ganglia forthe first time here (Table 1). Of the four sNPFs, two were detectedin multiple tissues (Table 1).

3.5.3. Other RFamidesNineteen amino (N)-terminally extended –FLRFamides were se-

quenced from the neural extracts of O. ceratophthalma (Table 1), 15from the brain, 10 each from the PO and eyestalk ganglia, and fourfrom the SG. Ten of the 19 identified peptides were found in multipletissues (Table 1). While the majority of the N-terminally extended –FLRFamides (16 of the 19 peptides) are previously known isoforms,e.g. RDRNFLRFamide and APRNFLRFamide from C. productus [19],three, AYNQSFLRFamide, NQPGVNFLRFamide and SVGNRNFLRFa-mide, were novel identifications, described here for the first time.

Fig. 1. Identification of neuropeptides in the brain of Ocypode ceratophthalma via MALDI-TOF/TOF accurate mass matching. (A) Detection of peptides in crude brain extract.(B) Detection of peptides in an HPLC separated fraction (Fraction 6) of brain extract.

Fig. 2. NanoLC-ESI-Q-TOF de novo sequencing of SPNDWAHFRGSWamide, a novelB-type allatostatin, from the pericardial organ using (A) Q-TOF Micromass or (B)SYNAPT G2 HDMS systems. In this and all other de novo sequencing figures, b-ionsrepresent m/z peaks where charge is maintained on amino (N)-terminal peptidefragments, while y-ions represent peaks where charge is maintained on carboxy(C)-terminal fragments.


Three FLPs possessing –YLRFamide C-termini were identifiedfrom O. ceratophthalma brain (2 peptides) or eyestalk ganglia (1peptide), including the novel isoform YGNKNYLRFamide se-quenced from eyestalk extracts (Table 1).

In addition to the –F/YLRFamides, two peptides possessing –NLRFamide C-termini, including the novel isoform NRSNLRFamide

(Figs. 1A, B and 5B), were sequenced from the eyestalk and/or brainof O. ceratophthalma (Table 1).

3.6. Orcokinin

Forty-seven peptides showing sequence homology to membersof the orcokinin family (full-length, mature peptides typicallybeing 13 amino acids long and possessing the N-terminal consen-sus motif NFDEIDR– [9]), were identified from the neural extractsof O. ceratophthalma (Table 1). Of these peptides, which includeputative full-length peptides, truncations and amidated isoforms,19 are novel, being identified for the first time here: NFDEIDR,DFDEIDR, NFDEIDRS, NFDEIDRSG, DFDEIDRSG, NFDEIDRSS,EIDRSSFGFH (Fig. 1B), NFDEIDRSSF, NFDEIDRSGFV, FDEIDRSSFGF,NFDEIDRSDFA, FDEIDRSGFGFV, FDEIDRSSFGFA, DFDEIDRSGFGF(Fig. 6A), DFDEIDRSSFGF, NFDEIDRSGFGFVamide, NFDEIDRS-GYGFV, NFDEIDRSSFGFH (Fig. 1A) and DFDEIDRSSFGFH. Theremaining peptides were previously described from other crusta-ceans, e.g. the amidated orcokinin variants NFDEIDRSGFamideand NFDEIDRSSFamide from H. americanus and C. maenas, respec-tively [36,37].

While orcokinins were detected in all the O. ceratophthalma tis-sues examined, the PO was a particularly rich source of members ofthis family, with 37 of the 47 peptides identified in this tissue (Ta-ble 1). Of the 47 orcokinins identified here, 24 were present in mul-tiple tissues (Table 1).

3.7. Orcomyotropin

Four peptides showing sequence homology to FDAFTTGFamide[15], commonly referred to as orcomyotropin [9], were identifiedfrom the neural extracts of O. ceratophthalma (Table 1). All four

Fig. 3. NanoLC-ESI-Q-TOF de novo sequencing of putative truncated crustaceanhyperglycemic hormone precursor-related peptides (CPRPs) from the sinus glandvia Q-TOF Micromass. (A) De novo sequencing of LPSAHPLE. (B) De novo sequencingof SLRGPAESSGESALSE. (C) De novo sequencing of SLRGPAESDGESAHPLE. (D) Denovo sequencing of LLTSLRGPAESSGESAHPLE.

Fig. 4. Sequence alignment of truncated crustacean hyperglycemic hormoneprecursor-related peptides (CPRPs) using the online software program MAFFTversion 6 (http://mafft.cbrc.jp/alignment/software/; [30,31]). Three of the tentruncations (CPRP-I, CPRP-II and CPRP-III) possess sequences that suggest theyare from CPRPs distinct from the remaining seven peptides (which appear to bederived from a common CPRP; CPRP-IV). In this figure, amino acids that arecommon to all truncations in their regions of overlap are highlighted in black, whileresidues that are shared by all but one peptide are highlighted in gray; variableresidues (as well as a single apparent deletion) are unhighlighted in this figure. Thenumbers in parentheses to the right of the CPRP-IVs show the number of amino acidresidues contained in the truncation.

Fig. 5. NanoLC-ESI-Q-TOF de novo sequencing of novel FMRFamide-like peptidesfrom the eyestalk ganglia via SYNAPT G2 HDMS. (A). De novo sequencing of theshort neuropeptide F isoform DVRAPALRLRFamide. (B) De novo sequencing ofNRSNLRFamide.

Fig. 6. NanoLC-ESI-Q-TOF de novo sequencing of novel orcokinin and orcomyotro-pin peptides. (A) De novo sequencing of the putative truncated orcokininDFDEIDRSGFGF from the pericardial organ via (A1) Q-TOF Micromass and (A2)SYNAPT G2 HDMS. (B) De novo sequencing of the orcomyotropin FDAYTTGFGHSfrom the eyestalk ganglia via SYNAPT G2 HDMS.


peptides were unamidated, C-terminally extended isoforms (Ta-ble 1), three being previously known variants, e.g. the broadly con-served FDAFTTGFGHS (e.g. [26,27,36–38,40,51]), and the fourth,

FDAYTTGFGHS (Fig. 6B), a novel isoform sequenced from the eye-stalk ganglia for the first time here. All tissues contained at leastone isoform of orcomyotropin, and two of the isoforms were pres-ent in multiple tissues (Table 1).

3.8. Orcokinin/orcomyotropin precursor-related peptide

In decapods, multiple orcokinins and one copy of orcomyotro-pin are encoded on a common precursor protein [12,60]. In

http://mafft.cbrc.jp/alignment/software/

Fig. 7. NanoLC-ESI-Q-TOF de novo sequencing of novel RYamide isoforms from thepericardial organ via SYNAPT G2 HDMS. (A) De novo sequencing of SGFYSNRYamide.(B) De novo sequencing of SGFYSDRYamide. (C) De novo sequencing ofSGFYCNRYamide.


addition, a number of precursor-related peptides (PRP) are en-coded with these peptides on the prepro-orcokinin/orcomyotropin[12,60]. Here, we have sequenced TPRDIANLY from the SG of O. cer-atophthalma (Table 1); this peptide is identical to an orcokinin/orcomyotropin precursor-related peptide recently described fromC. sapidus [26].

3.9. Pigment dispersing hormone

The peptide NSELINSILGLPKVMNDAamide, commonly referredto as b-pigment dispersing hormone or b-PDH [9], was identifiedfrom the brain, eyestalk ganglia and SG of O. ceratophthalma (Ta-ble 1); this peptide has previously been identified from a wide vari-ety of decapod species, including a number of brachyurans (e.g.[19,23,26,32,36,40,46]). In addition, three novel, putative trunca-tions of b-PDH (one from the SG and two from the eyestalk ganglia;Table 1), and a novel oxidized version of this peptide (from thebrain and SG; Table 1), were sequenced here for the first time.

3.10. Proctolin

The peptide RYLPT, commonly referred to as proctolin [9], wasdetected in all of the O. ceratophthalma tissues examined in thisstudy (Table 1); proctolin has previously been identified from awide variety of crustaceans (e.g. [19,26,27,33,36–38,40,47,59]).

3.11. Pyrokinin

Two peptides exhibiting the C-terminal motif –FXPRLamide(where X is a variable residue), the hallmark of the pyrokinin fam-ily [9], were identified in O. ceratophthalma (Table 1). These pep-tides, LYFAPRLamide (detected in the eyestalk ganglia) andDTGFAFSPRLamide (identified in the PO), are known crustaceanpyrokinins, with the former previously sequenced from C. borealis[40] and the latter a known C. maenas isoform [36].

3.12. RYamide

Thirteen peptides possessing the C-terminal motif –RYamide,the hallmark of the RYamide family [9], were identified in O. cerat-ophthalma (Table 1). Of these peptides, five, SGFYALRYamide,SGFYSNRYamide (Fig. 7A), SGFYSDRYamide (Fig. 7B), SGFYCNRYa-mide (Fig. 7C) and SGFNSPSPRYamide, are novel isoforms, se-quenced here for the first time. The remaining eight RYamidesare known crustacean isoforms, e.g. the broadly conserved pEG-FYSQRYamide (Fig. 1A and B) [33,55].

The PO was by far the richest source of RYamides, with all 13isoforms present in this tissue (Table 1). In contrast, only threeRYamides were identified in the brain, with but a single isoformeach present in the eyestalk ganglia and SG (Table 1). Only threeof the 13 RYamides were present in multiple tissues (Table 1).

3.13. SIFamide

Two peptides exhibiting the C-terminal motif –SIFamide, thehallmark of the SIFamide family [9], were identified in O. cerat-ophthalma: GYRKPPFNGSIFamide (Fig. 1A) and RKPPFNGSIFamide(Table 1). Both peptides are known, broadly conserved crustaceanisoforms (e.g. [49,56]), with the former detected in brain, eyestalkganglia and SG extracts and the latter identified from brain only(Table 1).

3.14. Tachykinin-related peptide

Seven peptides possessing the C-terminal motif –FXGXRamide(where the two Xs represent variable amino acids), the hallmark

of the tachykinin-related peptide (TRP) family [9], were identifiedfrom O. ceratophthalma tissue extracts (Table 1). All of the TRPs ap-pear to be related to the mature, full-length isoforms APS-GFLGMRamide or TPSGFLGMRamide, with one partiallyprocessed, two oxidized, and two truncated versions of them in-cluded in the collection of peptides identified from O. ceratophth-alma (Table 1). APSGFLGMRamide (Fig. 1A and B) orTPSGFLGMRamide (Fig. 1A and B) are previously known crustaceanTRPs (e.g. [7,56,58]), and the partially processed, oxidized and trun-cated versions of them have also been previously described fromother decapods (e.g. [7,37,58]).

All of the sequenced TRPs were present in extracts of the eye-stalk ganglia, with six of the seven also sequenced from the brain(Table 1). Three of the seven TRPs were detected in the SG, withtwo sequenced from the PO (Table 1). All of the TRPs were detectedin at least two of the four tissues sampled in our study (Table 1).

3.15. Other peptides

In addition to members of the families described above, twoother peptides were identified here from the neural extracts of O.ceratophthalma: HIGSLYRamide (from the brain [Fig. 1A and Ta-ble 1], eyestalk ganglia and SG) and HIGSLLRamide (from the eye-stalk ganglia only [Table 1]). HIGSLYRamide is a known crustaceanneuropeptide, having originally been identified from C. productus[19]; HIGSLLRamide is described here for the first time.

4. Discussion

4.1. The neuropeptidome of Ocypode ceratophthalma

In the study presented here, MALDI-TOF/TOF MS and nanoLC-ESI-Q-TOF MS/MS were used to elucidate the neuropeptidome of


the crab O. ceratophthalma. In total 156 peptides were identifiedusing this combined approach, of which 59 were novel, being de-scribed here for the first time. The peptides identified includedmembers of 15 families, and the collection, in its totality, repre-sents the largest number of peptides thus far characterized viamass spectrometry from any crustacean in a single study.

Members of the orcokinin family were by far the largest singlegroup of peptides identified from O. ceratophthalma, with 47 pep-tides characterized. In addition, a large number of FLPs, 30 pep-tides, were identified from the neural tissues of this species.Interestingly, several highly conserved peptides/peptide familiesthat are present in the peptidomes of other decapods went unde-tected in our study, including members of the crustacean hypergly-cemic hormone (CHH) superfamily (i.e. CHH, molt-inhibitinghormone [MIH] and mandibular organ-inhibiting hormone[MOIH]), the C-type allatostatins (C-ASTs), corazonin, and red pig-ment concentrating hormone (RPCH). It is not surprising that wedid not characterize isoforms of CHH, MIH or MOIH, as these pep-tides are typically over 70 amino acids in length [9], too long to beeffectively sequenced using the methodology/MS platforms em-ployed here. In contrast, the known, and highly conserved, decapodC-type ASTs (pQIRYHQCYFNPISCF and SYWKQCAFNAVSCFamide),corazonin (pQTFQYSRGWTNamide) and RPCH (pELNFSPGWa-mide), see [9] for a recent review of these peptides in decapod spe-cies, should have been detectable via the methods we used.

Several possibilities exist for our failure to identify the C-ASTs,corazonin and RPCH in O. ceratophthalma. First, it is possible thatthey are simply absent in the tissues surveyed. This seems unlikelyas previous mass spectral work has identified both C-type AST pep-tides in the brain and PO of a number of crustacean species, includ-ing brachyurans [14,39,54]. Likewise, corazonin has previouslybeen detected via MS in crustacean brains and/or POs[33,36,38,40], with RPCH commonly found via MS in SGs[13,19,36,37,40]. Moreover, immunohistochemistry suggests thepresence of at least corazonin-like and RPCH-like immunoreactiveneurons in the brain of O. ceratophthalma (A.E. Christie, unpub-lished). More likely is that these peptides are present in O. cerat-ophthalma, but in amounts that are below our levels of detection,or that signals derived from their ionized forms have been sup-pressed by other peptides/compounds present in the analyzed ex-tracts. Alternatively, it is possible that the native O. ceratophthalmaisoforms of C-AST, corazonin and/or RPCH differ in amino acidcomposition and/or post-translational modification from those ofother decapods. Clearly, additional study will be needed to clarifythis issue.

4.2. Relative contributions of different mass spectral platforms to theelucidation of a neuropeptidome

In this study, a mass spectrometric strategy combining MALDI-TOF/TOF MS and nanoLC-ESI-Q-TOF MS/MS was used to elucidatethe neuropeptidome of O. ceratophthalma. Specifically, the highlysensitive and accurate mass measurements provided by MALDI-TOF/TOF MS were used to identify known peptides based on theirmass-to-charge ratios (m/z), while nanoLC-ESI-Q-TOF MS/MS wasemployed to de novo sequence peptides from the O. ceratophthalmanervous system. These platforms, while using different ionizationmethods, are complementary to one another and, in combinationwith advanced separation technology, enable the discovery of amaximum number of neuropeptides. For example, 35 peptideswere detected in the brain using MALDI-TOF/TOF MS, 53 wereidentified from this tissue by combining the results from Micro-mass and SYNAPT G2 ESI-Q-TOF MS/MS; 16 brain peptides weredetected by both platforms.

Off-line HPLC separation greatly simplifies the chemical com-plexity of biological matrices, such as crude neural tissue extracts,

by removing salts, lipids and proteins that can interfere with theionization efficiency of neuropeptides, the targets of the study pre-sented here. For example, we were able to detect only 17 neuro-peptides via MALDI-TOF/TOF MS in crude extract of the O.ceratophthalma brain, whereas 35 were revealed after the extractwas subjected to HPLC separation. This finding highlights theadvantage of incorporating separation techniques to reduce sam-ple complexity and increase dynamic range. In addition, the off-line HPLC can be coupled to a nanoLC, which is connected directlyto the ESI-Q-TOF, and serves as an additional dimension in 2D-LCfor further separation of neuropeptides. Again, the combinationof microscale separation methods and complementary mass spec-tral techniques provides enhanced neuropeptidome coverage.

Two ESI-Q-TOF mass spectrometers were used in this study, aQ-TOF Micromass and a SYNAPT G2 HDMS. While the principlesgoverning the function of these two instruments are the same, dif-ferent setup and detection parameters make the peptide fragmen-tation patterns generated from these two instruments distinct. Forexample, the Q-TOF Micromass instrument produced more intensefragment ions in the low mass range than did the SYNAPT G2HDMS system, and therefore provided more complete informationon immonium ions, which are indicative of a peptide’s amino acidcomposition. In contrast, the SYNAPT G2 HDMS provided highersensitivity and better coverage than did the Q-TOF Micromassmass spectrometer, as the ion optics and resolution have been im-proved in this new generation instrument. Moreover, the SYNAPTG2 provided more information on large fragment ions than didthe Q-TOF Micromass system, which helped in the elucidation ofpeptide sequence structure. Additionally, for some peptide fami-lies, e.g. the orcokinins, more b-type ions are detected using theSYNAPT G2 instrument; typically only a series of y-type fragmentions at high mass range were detected for these peptides usingthe Q-TOF Micromass mass spectrometer, which makes deductionof peptide sequences more difficult. Thus, the complementaryinformation provided by the Q-TOF Micromass and SYNAPT G2HDMS instruments greatly improved our ability for ESI-Q-TOF denovo sequencing.

4.3. Potential uses of Ocypode ceratophthalma peptidomic data

As stated earlier, the horn-eyed ghost crab O. ceratophthalma isprimarily a terrestrial species [22]. Thus, its modulation of physio-logical processes like water and ion transport, respiration, etc., maybe under quite different hormonal control from freshwater andmarine species. The catalog of neuropeptides identified here pro-vides a foundation for future investigation of the hormonal controlof these and other processes in this terrestrial decapod species.Interestingly, ghost crabs are often billed as among the fastest landcrustaceans [20]; they also construct elaborate burrows using theirchelipeds and walking legs [22]. These behaviors are undoubtedlycontrolled by the thoracic nervous system, which provides inner-vation to the leg musculature. The catalog of peptides identifiedhere now allows for investigation of how these molecules are ableto modulate/integrate the distinct and complex movements neces-sary for enabling these behaviors. Finally, ghost crabs have longbeen known to exhibit robust circadian rhythms in locomotion[3] and color change [35]. The catalog of peptides presented herenow positions us to begin to assess which of, and how, these mol-ecules may function as output signals from the circadian system toaffect these and other daily rhythms in physiology and behavior.

Acknowledgments

We thank Dr. Ian Cooke for providing us access to lodging and abase for field collection and dissection at Malaekahana Beach. Wealso thank Dr. Daniel Hartline, Dr. Petra Lenz, Nico Hartline, Kara


Chin, and Christina Linkem for their help in collecting some of theanimals used in this study, and Tiana Fontanilla for reading andcommenting on early drafts of this manuscript. Support for thisstudy was provided by the National Science Foundation (CHE-0957784 to L.L.), the National Institutes of Health(1R01DK071801 to L.L.) and the Cades Foundation of Honolulu, Ha-waii (to A.E.C).

References

[1] D.E. Bliss, Metabolic effects of sinus gland or eyestalk removal in the land crab,Gecarcinus lateralis, Anat. Rec. 111 (1951) 502–503.

[2] R.C. Brusca, G.J. Brusca, Invertebrates, 2nd ed., Sinauer Associates, Sunderland,MA, 2003.

[3] M. Burrows, G. Hoyle, The mechanism of rapid running in the ghost crab,Ocypode ceratophthalma, J. Exp. Biol. 58 (1973) 327–349.

[4] S.S. Cape, K.J. Rehn, M. Ma, E. Marder, L. Li, Mass spectral comparison of theneuropeptide complement of the stomatogastric ganglion and brain in theadult and embryonic lobster, Homarus americanus, J. Neurochem. 105 (2008)609–702.

[5] R. Chen, X. Jiang, M.C. Conaway, I. Mohtashemi, L. Hui, R. Viner, L. Li, Massspectral analysis of neuropeptide expression and distribution in the nervoussystem of the lobster Homarus americanus, J. Proteome Res. 9 (2010) 818–832.

[6] A.E. Christie, Crustacean neuroendocrine systems and their signaling agents,Cell Tissue Res. 345 (2011) 41–67.

[7] A.E. Christie, C.T. Lundquist, D.R. Nässel, M.P. Nusbaum, Two novel tachykinin-related peptides from the nervous system of the crab Cancer borealis, J. Exp.Biol. 200 (1997) 2279–2294.

[8] A.E. Christie, M.D. McCoole, From genes to behavior: investigations ofneurochemical signaling come of age for the model crustacean Daphniapulex, J. Exp. Biol. 215 (2012) 2535–2544.

[9] A.E. Christie, E.A. Stemmler, P.S. Dickinson, Crustacean neuropeptides, Cell Mol.Life Sci. 67 (2010) 4135–4169.

[10] J.S. Chung, D.C. Wilcockson, N. Zmora, Y. Zohar, H. Dircksen, S.G. Webster,Identification and developmental expression of mRNAs encoding crustaceancardioactive peptide (CCAP) in decapod crustaceans, J. Exp. Biol. 209 (2006)3862–3872.

[11] I.M. Cook, Reliable, responsive pacemaking and pattern generation withminimal cell numbers: the crustacean cardiac ganglion, Biol. Bull. 202 (2002)108–136.

[12] P.S. Dickinson, E.A. Stemmler, E.E. Barton, C.R. Cashman, N.P. Gardner, S. Rus,H.R. Brennan, T.S. McClintock, A.E. Christie, Molecular, mass spectral, andphysiological analyses of orcokinins and orcokinin precursor-related peptidesin the lobster Homarus americanus and the crayfish Procambarus clarkii,Peptides 30 (2009) 297–317.

[13] P.S. Dickinson, E.A. Stemmler, A.E. Christie, The pyloric neural circuit of theherbivorous crab Pugettia producta shows limited sensitivity to severalneuromodulators that elicit robust effects in more opportunistically feedingdecapods, J. Exp. Biol. 211 (2008) 1434–1447.

[14] P.S. Dickinson, T. Wiwatpanit, E.R. Gabranski, R.J. Ackerman, J.S. Stevens, C.R.Cashman, E.A. Stemmler, A.E. Christie, Identification ofSYWKQCAFNAVSCFamide: a broadly conserved crustacean C-typeallatostatin-like peptide with both neuromodulatory and cardioactiveproperties, J. Exp. Biol. 212 (2009) 1140–1152.

[15] H. Dircksen, S. Burdzik, A. Sauter, R. Keller, Two orcokinins and the noveloctapeptide orcomyotropin in the hindgut of the crayfish Orconectes limosus:identified myostimulatory neuropeptides originating together in neurones ofthe terminal abdominal ganglion, J. Exp. Biol. 203 (2000) 2807–2818.

[16] V. Fénelon, Y. Le Feuvre, T. Bem, P. Meyrand, Maturation of rhythmic neuralnetworks: role of central modulatory inputs, J. Physiol. Paris. 97 (2003) 59–68.

[17] P. Fernlund, L. Josefsson, Crustacean color-change hormone: amino acidsequence and chemical synthesis, Science 177 (1972) 173–175.

[18] Q. Fu, A.E. Christie, L. Li, Mass spectrometric characterization of crustaceanhyperglycemic hormone precursor-related peptides (CPRPs) from the sinusgland of the crab, Cancer productus, Peptides 26 (2005) 2137–2150.

[19] Q. Fu, K.K. Kutz, J.J. Schmidt, Y.W. Hsu, D.I. Messinger, S.D. Cain, H.O. de laIglesia, A.E. Christie, L. Li, Hormone complement of the Cancer productus sinusgland and pericardial organ: an anatomical and mass spectrometricinvestigation, J. Comp. Neurol. 493 (2005) 607–626.

[20] D.R. Hafemann, J.I. Hubbard, On the rapid running of ghost crabs (OcypodeCeratophthalma), J. Exp. Zool. 170 (1969) 25–31.

[21] S.L. Hooper, R.A. DiCaprio, Crustacean motor pattern generator networks,Neurosignals 13 (2004) 50–69.

[22] J.P. Hoover, Hawaii’s Sea Creatures, Revised Edition., Mutual Publishing LLC,Honolulu, HI, 2010.

[23] Y.W. Hsu, E.A. Stemmler, D.I. Messinger, P.S. Dickinson, A.E. Christie, H.O. de laIglesia, Cloning and differential expression of two b-pigment-dispersinghormone (b-PDH) isoforms in the crab Cancer productus: evidence forauthentic b-PDH as a local neurotransmitter and b-PDH II as a humoralfactor, J. Comp. Neurol. 508 (2008) 197–211.

[24] Y.W. Hsu, J.R. Weller, A.E. Christie, H.O. de la Iglesia, Molecular cloning of fourcDNAs encoding prepro-crustacean hyperglycemic hormone (CHH) from theeyestalk of the red rock crab Cancer productus: identification of two genetically

encoded CHH isoforms and two putative post-translationally derived CHHvariants, Gen. Comp. Endocrinol. 155 (2008) 517–525.

[25] A.E. Hudson, S. Archila, A.A. Prinz, Identifiable cells in the crustaceanstomatogastric ganglion, Physiology (Bethesda) 25 (2010) 311–318.

[26] L. Hui, R. Cunningham, Z. Zhang, W. Cao, C. Jia, L. Li, Discovery andcharacterization of the Crustacean hyperglycemic hormone precursor relatedpeptides (CPRP) and orcokinin neuropeptides in the sinus glands of the bluecrab Callinectes sapidus using multiple tandem mass spectrometry techniques,J. Proteome Res. 10 (2011) 4219–4229.

[27] L. Hui, F. Xiang, Y. Zhang, L. Li, Mass spectrometric elucidation of theneuropeptidome of a crustacean neuroendocrine organ, Peptides 36 (2012)230–239.

[28] L. Hui, Y. Zhang, J. Wang, A. Cook, H. Ye, M.P. Nusbaum, L. Li, Discovery andfunctional study of a novel crustacean tachykinin neuropeptide, ACS Chem.Neurosci. 2 (2011) 711–722.

[29] J. Huybrechts, M.P. Nusbaum, L.V. Bosch, G. Baggerman, A. De Loof, L. Schoofs,Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonahcrab, Cancer borealis, Biochem. Biophys. Res. Commun. 308 (2003) 535–544.

[30] K. Katoh, K. Misawa, K. Kuma, T. Miyata, MAFFT: a novel method for rapidmultiple sequence alignment based on fast Fourier transform, Nucleic AcidsRes. 30 (2002) 3059–3066.

[31] K. Katoh, H. Toh, Recent developments in the MAFFT multiple sequencealignment program, Brief Bioinform. 9 (2008) 286–298.

[32] J.M. Klein, C.J. Mohrherr, F. Sleutels, J.P. Riehm, K.R. Rao, Molecular cloning oftwo pigment-dispersing hormone (PDH) precursors in the blue crab Callinectessapidus reveals a novel member of the PDH neuropeptide family, Biochem.Biophys. Res. Commun. 205 (1994) 410–416.

[33] L. Li, W.P. Kelley, C.P. Billimoria, A.E. Christie, S.R. Pulver, J.V. Sweedler, E.Marder, Mass spectrometric investigation of the neuropeptide complementand release in the pericardial organs of the crab, Cancer borealis, J. Neurochem.87 (2003) 642–656.

[34] L. Li, J.V. Sweedler, Peptides in the brain: measurement approaches andchallenges, Annu. Rev. Anal. Chem. 1 (2008) 451–483.

[35] G. Little, Chromatophore responses in relation to the photoperiod andbackground color in the Hawaiian ghost crab, Ocypode ceratophthalma(Pallas), Pacific Sci. 21 (1967) 77–84.

[36] M. Ma, E.K. Bors, E.S. Dickinson, M.A. Kwiatkowski, G.L. Sousa, R.P. Henry, C.M.Smith, D.W. Towle, A.E. Christie, L. Li, Characterization of the Carcinus maenasneuropeptidome by mass spectrometry and functional genomics, Gen. Comp.Endocrinol. 161 (2009) 320–334.

[37] M. Ma, R. Chen, G.L. Sousa, E.K. Bors, M.A. Kwiatkowski, C.C. Goiney, M.F. Goy,A.E. Christie, L. Li, Mass spectral characterization of peptide transmitters/hormones in the nervous system and neuroendocrine organs of the Americanlobster Homarus americanus, Gen. Comp. Endocrinol. 156 (2008) 395–409.

[38] M. Ma, A.L. Gard, F. Xiang, J. Wang, N. Davoodian, P.H. Lenz, S.R. Malecha, A.E.Christie, L. Li, Combining in silico transcriptome mining and biological massspectrometry for neuropeptide discovery in the Pacific white shrimpLitopenaeus vannamei, Peptides 31 (2010) 27–43.

[39] M. Ma, T.M. Szabo, C. Jia, E. Marder, L. Li, Mass spectrometric characterizationand physiological actions of novel crustacean C-type allatostatins, Peptides 30(2009) 1660–1668.

[40] M. Ma, J. Wang, R. Chen, L. Li, Expanding the crustacean neuropeptidome usinga multifaceted mass spectrometric approach, J. Proteome Res. 8 (2009) 2426–2437.

[41] E. Marder, D. Bucher, Understanding circuit dynamics using the stomatogastricnervous system of lobsters and crabs, Ann. Rev. Physiol. 69 (2007) 291–316.

[42] M.P. Nusbaum, M.P. Beenhakker, A small-systems approach to motor patterngeneration, Nature 417 (2002) 343–350.

[43] M.P. Nusbaum, D.M. Blitz, Neuropeptide modulation of microcircuits, Curr.Opin. Neurobiol. 22 (2012) 592–601.

[44] M.P. Nusbaum, D.M. Blitz, A.M. Swensen, D. Wood, E. Marder, The role of co-transmission in neural network modulation, Trends Neurosci. 24 (2001) 146–154.

[45] L.M. Passano, The X-organ-sinus gland system neurosecretory system of crabs,Anat. Rec. 111 (1951) 502.

[46] K.R. Rao, J.P. Riehm, C.A. Zahnow, L.H. Kleinholz, G.E. Tarr, L. Johnson, S. Norton,M. Landau, O.J. Semmes, R.M. Sattelberg, W.H. Jorenby, M.F. Hintz,Characterization of a pigment-dispersing hormone in eyestalks of the fiddlercrab Uca pugilator, Proc. Natl. Acad. Sci. USA 82 (1985) 5319–5322.

[47] T.L. Schwarz, G.M. Lee, K.K. Siwicki, D.G. Standaert, E.A. Kravitz, Proctolin in thelobster: the distribution, release, and chemical characterization of a likelyneurohormone, J. Neurosci. 4 (1984) 1300–1311.

[48] A.I. Selverston, J. Ayers, Oscillations and oscillatory behavior in small neuralcircuits, Biol. Cybern. 95 (2006) 537–554.

[49] P. Sithigorngul, J. Pupuem, C. Krungkasem, S. Longyant, P. Chaivisuthangkura,W. Sithigorngul, A. Petsom, Seven novel FMRFamide-like neuropeptidesequences from the eyestalk of the giant tiger prawn Penaeus monodon,Comp. Biochem. Physiol. B Biochem. Mol. Biol. 131 (2002) 325–337.

[50] P. Skiebe, Neuropeptides are ubiquitous chemical mediators: using thestomatogastric nervous system as a model system, J. Exp. Biol. 204 (2001)2035–2048.

[51] P. Skiebe, M. Dreger, J. Börner, M. Meseke, W. Weckwerth,Immunocytochemical and molecular data guide peptide identification bymass spectrometry: orcokinin and orcomyotropin-related peptides in thestomatogastric nervous system of several crustacean species, Cell Mol. Biol.(Noisy-le-grand) 49 (2003) 851–871.


[52] J. Stangier, C. Hilbich, K. Beyreuther, R. Keller, Unusual cardioactive peptide(CCAP) from pericardial organs of the shore crab Carcinus maenas, Proc. Natl.Acad. Sci. USA 84 (1987) 575–579.

[53] W. Stein, Modulation of stomatogastric rhythms, J. Comp. Physiol. ANeuroethol. Sens. Neural Behav. Physiol. 195 (2009) 989–1009.

[54] E.A. Stemmler, E.A. Bruns, C.R. Cashman, P.S. Dickinson, A.E. Christie, Molecularand mass spectral identification of the broadly conserved decapod crustaceanneuropeptide pQIRYHQCYFNPISCF: the first PISCF-allatostatin (Manduca sexta- orC-type allatostatin) from a non-insect, Gen. Comp. Endocrinol. 165 (2010) 1–10.

[55] E.A. Stemmler, E.A. Bruns, N.P. Gardner, P.S. Dickinson, A.E. Christie, Massspectrometric identification of pEGFYSQRYamide: a crustacean peptidehormone possessing a vertebrate neuropeptide Y (NPY)-like carboxy-terminus, Gen. Comp. Endocrinol. 152 (2007) 1–7.

[56] E.A. Stemmler, C.R. Cashman, D.I. Messinger, N.P. Gardner, P.S. Dickinson, A.E.Christie, High-mass-resolution direct-tissue MALDI-FTMS reveals broadconservation of three neuropeptides (APSGFLGMRamide, GYRKPPFNGSIFamide and pQDLDHVFLRFamide) across members of seven decapodcrustaean infraorders, Peptides 28 (2007) 2104–2115.

[57] E.A. Stemmler, Y.W. Hsu, C.R. Cashman, D.I. Messinger, H.O. de la Iglesia, P.S.Dickinson, A.E. Christie, Direct tissue MALDI-FTMS profiling of individualCancer productus sinus glands reveals that one of three distinct combinationsof crustacean hyperglycemic hormone precursor-related peptide (CPRP)isoforms are present in individual crabs, Gen. Comp. Endocrinol. 154 (2007)184–192.

[58] E.A. Stemmler, B. Peguero, E.A. Bruns, P.S. Dickinson, A.E. Christie,Identification, physiological actions, and distribution of TPSGFLGMRamide: anovel tachykinin-related peptide from the midgut and stomatogastric nervoussystem of Cancer crabs, J. Neurochem. 101 (2007) 1351–1366.

[59] R.E. Sullivan, A proctolin-like peptide in crab pericardial organs, J. Exp. Zool.210 (1979) 543–552.

[60] Y. Yasuda-Kamatani, A. Yasuda, Identification of orcokinin gene-relatedpeptides in the brain of the crayfish Procambarus clarkii by the combinationof MALDI-TOF and on-line capillary HPLC/Q-TOF mass spectrometries andmolecular cloning, Gen. Comp. Endocrinol. 118 (2000) 161–172.

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