Venomics of Remipede Crustaceans Reveals Novel Peptide Diversity and Illuminates the Venom's ...

25 Pages·2017·7.76 MB·English

toxins
Article
Venomics of Remipede Crustaceans Reveals Novel
Peptide Diversity and Illuminates the Venom’s
Biological Role
BjörnM.vonReumont1,2,*,EivindA.B.Undheim3,*,Robin-TobiasJauss1andRonaldA.Jenner2,*
1 MolecularEvolutionandSystematicsofAnimals,InstituteforBiology,UniversityofLeipzig,
Leipzig04103,Germany;[email protected]
2 DepartmentofLifeSciences,NaturalHistoryMuseum,CromwellRoad,LondonSW75BD,UK
3 CentreforAdvancedImaging,UniversityofQueensland,St.Lucia4072,QLD,Australia
* Correspondence:[email protected](B.M.v.R.);[email protected](E.A.B.U.);[email protected](R.A.J.);
Tel.:[phone](B.M.v.R.);[phone](E.A.B.U.);[phone](R.A.J.)
AcademicEditors:NicholasR.CasewellandSyedA.Ali
Received:27June2017;Accepted:24July2017;Published:26July2017
Abstract: Wereportthefirstintegratedproteomicandtranscriptomicinvestigationofacrustacean
venom. Remipedecrustaceansarethevenomoussistergroupofhexapods,andthevenomglands
of the remipede Xibalbanus tulumensis express a considerably more complex cocktail of proteins
andpeptidesthanpreviouslythought. Weidentified32venomproteinfamilies,including13novel
peptide families that we name xibalbins, four of which lack similarities to any known structural
class. Our proteomic data confirm the presence in the venom of 19 of the 32 families. The most
highly expressed venom components are serine peptidases, chitinase and six of the xibalbins.
The xibalbins represent Inhibitory Cystine Knot peptides (ICK), a double ICK peptide, peptides
withaputativeCystine-stabilizedα-helix/β-sheetmotif,apeptidesimilartohairpin-likeβ-sheet
forming antimicrobial peptides, two peptides related to different hormone families, and four
peptides with unique structural motifs. Remipede venom components represent the full range
ofevolutionaryrecruitmentfrequencies,fromfamiliesthathavebeenrecruitedintomanyanimal
venoms(serinepeptidases,ICKs),tothosehavingaverynarrowtaxonomicrange(doubleICKs),
tothoseuniqueforremipedes. Wediscussthemosthighlyexpressedvenomcomponentstoshed
lightontheirpossiblefunctionalsignificanceinthepredatoryanddefensiveuseofremipedevenom,
andtoprovidetestableideasforanyfuturebioactivitystudies.
Keywords: venomics;Remipedia;crustaceans;ICK;venom;arthropods;anchialinecaves
1. Introduction
Remipedesaresuperficiallycentipede-likepredatorycrustaceansthatexclusivelyinhabitmarine
cavesystems[1]. Therearecurrently29describedspecies[2],andtheirfirstdescriptionin1981[3]
rekindledtheinterestofzoologiststoattempttoreconstructtheevolutionaryoriginofcrustaceans.
Carcinologists were quick to interpret remipedes’ long segmented bodies, their lack of segment
specialization,theirlackofacarapace,andtheirseriallyhomonomous,biramous,andpaddle-like
trunklimbsasevidencethattheycouldwellbetheearliestdiverginglineageofextantcrustaceans
withthemostprimitivebodyplan[4,5]. Sincethenmolecularevidencehaspaintedaratherdifferent
picture. Phylogenomic analyses suggest that remipedes are nested deeply within pancrustaceans
(thecladecomprisinginsectsandcrustaceans), asthelivingsistergroupofinsects[6–9]. Thishas
drawnremipedesintodebatesabouttheoriginofthemostsuccessfulgroupofanimalsonEarth[10],
but it also diminishes their relevance for understanding early crustacean evolution. At the same
Toxins2017,9,234;doi:10.3390/toxins9080234 www.mdpi.com/journal/toxins
Toxins2017,9,234 2of25
timethemolecularevidencerefocusesattentionontheremipedes’moststrikinglyuniquetrait: their
venomsystem.
ThevastmajorityofvenomousspeciesonEartharearthropods. Threeofthefourmaingroupsof
arthropods—chelicerates,myriapodsandinsects—togetherrepresentmorethan150,000venomous
species, and there are venomic studies for multiple species within each of these groups. In sharp
contrast,althoughputativelyvenomouscrustaceansexist,inparticularseveralparasiticspeciesof
copepods,malacostracansandbranchiurans[11],thevenomsystemofonlyasinglecrustaceanspecies
hasbeenthesubjectofatranscriptomicstudy[12].ThisshowedthattheremipedeXibalbanustulumensis
(previouslySpeleonectestulumensis)[13]isthefirstknownvenomouspredatorycrustacean. Remipede
biology and the environment in which they live provide some clues as to why remipedes have
evolvedvenom.
Withtheexceptionoftwospeciesthatoccurinfullymarine,sub-seafloorcaves[2],remipedes
areknownonlyfromanchialinecaves,whicharesubterraneancavesthatmergewiththeseaatthe
coastandgroundwaterinland. Theycontainalayeroffreshorbrackishwaterthatoverlaysalayerof
seawaterseparatedbyahalocline. Remipedesliveexclusivelyintheoxygenpoorsaltwaterzonesof
thesecaves,wherepreyabundanceislow. Remipedeslackeyes,buthavewell-developedolfactory
pathways[14],andtheyrelyonolfactorycuestonavigatetheirenvironment. Theyareactivepredators
ofcavecrustaceans,butarereportedasnormallybeingrelativelyslowswimmersthatarelikelytobe
outmanoeuvredbypotentialprey[15,16]. Undertheseconditionsitisadvantageoustopossessvenom
thatcanrapidlydebilitateprey. Remipedesmaythereforehaveevolvedavenomsystemconcomitant
withamoveintotheanchialineenvironment,andtheadoption,orelaboration,ofapredatorylifestyle
thatprimarilytargetsothercrustaceansthatinhabitthesamecavesystems.
InourpreviousstudyofX.tulumensiswediscoveredthatitsvenomapparatusismorphologically
morecomplexthanexpected,andcanfunctionasasophisticatedvenominjectionmechanism[12].
Their venom glands express a cocktail of transcripts that code for homologues of known venom
toxinsinothertaxa,withenzymesbeingthedominantcomponent. Thetranscriptsthatarethemost
highlyexpressedaswellasthemostdiversecodeforserinepeptidasesoftheS1familyandchitinases.
Themosthighlyexpressedtranscriptsfornon-enzymaticproteinscodeforcysteine-richpeptideswith
aputativeinhibitorcystineknot(ICK)motif,withsequencesimilaritiestospiderβ/δagatoxins.
Thestrikingdifferencesbetweenthistranscriptomicprofileandthevenomcompositionofother
venomousarthropophagousarthropodsareintriguing. Thevenomsofpredatoryarthropods,suchas
centipedes,scorpions,spiders,aswellasseveralhymenopterans,aretypicallyrichinpeptides[17–24],
withlargerproteinsandenzymesbeinglessabundant. Mostofthesepeptidesarethoughttohave
aroleinsubduingprey,andmanyactasparalyticneurotoxins. Thedearthoftranscriptscodingfor
peptides in the venom glands of X. tulumensis was therefore surprising. This apparent difference
invenomcompositioncouldbereal,andreflectdifferencesintheevolutionaryhistoryandbiology
ofthedifferentvenoms,butitcouldalsobeanartefactresultingfromthemethodologicalstrategy
of our original study. Bioinformatically identifying peptides in de novo assembled transcriptome
data from an unstudied organism in the absence of proteomic data is likely to underestimate the
truediversityofvenompeptides. Moreover,ourtranscriptomedataweregeneratedonthe454FLX
platformsequencingtechnology,whichdoesnotprovidethegreatestpossiblesequencingdepth.
Totestifthisapparentuniquenessofremipedevenomcompositionisrealorduetoinevitable
methodologicallimitationsofourinitialstudywe: (1)conductedaproteomicanalysisofthevenomof
theremipedeX.tulumensis;(2)producedanewtranscriptomeforthevenomglandsofX.tulumensis
usingIlluminaMiSeqtechnologybasedonthesameRNAmaterialusedforourprevious454FLX
analysis;and(3)re-sequencedthewholeanimaltranscriptomelibraryofX.tulumensiswithIllumina
HiSeqtechnology.
Toxins2017,9,234 3of25
2. ResultsandDiscussion
2.1. TheEffectofTranscriptomeSequencingPlatformsandAssemblyStrategies
2.1.1. TranscriptomeAssemblyStrategy
Contig sequences generated with SOAPdenovo-Trans and Trinity were generally identical,
althoughforsomeproteinclassesthedifferentassemblymethodsgeneratedslightlydifferentcontig
numbers. However, a comparison of these assembly strategies revealed that, on average, Trinity
assembliescontainmoresequencesthatBLASTtosecretedandputativetoxinproteins, aswellas
generallylongersequences(seeSupplementaryFigureS1)thantheSOAPdenovo-Transassemblies
(seeTable1). Consequently,wedecidedtousetheTrinityassembleddataforallfurtherdownstream
analyses(seealsoSection4.2).
Table 1. Overview of transcriptome libraries and assembly sizes (reads and numbers of contig
sequences)fordifferentassemblystrategies.Underlinednumbershighlighttheassemblystrategies
thatwereusedtoidentifyputativetoxinsfordownstreamanalyses.
RawData ClippedData CleanedData NumberofSecretedProteins
AssemblyStrategy
“Raw”Contigs Contigs>137nuc TrimmomaticSeqclean BLASTXvs.UniProt(SL0243)
Venomglandtranscriptome(library:27,421,129reads,pairedend)
SOAPdenovo-Trans(k-mer31) 294,931 177,668 176,408 -
SOAPdenovo-Trans(k-mer47) 247,453 198,075 197,240 17991
SOAPdenovo-Trans(k-mer65) 203.964 179,168 178,835 -
Trinity(readlength>101) 191,255 166,309 165,333 19431
Bodytranscriptome(library:9,165,598reads,pairedend)
SOAPdenovo-Trans(k-mer47) 151,399 123,676 123,241 23761
Trinity(readlength>101) 203,113 161,511 161,100 30041
1SeealsomethodsSection4.2.3.
2.1.2. Comparisonof454FLXandIlluminaMiSeqandHiSeqTranscriptomes
TheIlluminaMiSeqvenomglandtranscriptomeproduced105timesmoresequencereadsthan
the454FLXtranscriptomeinouroriginalstudy[12](27,421,129versus260,172reads),whichwere
assembledinto157timesmorecontigsinthepreferredTrinityassembly(197,240versus1052contigs).
TheIlluminaHiSeqwholebodytranscriptomeproducedninetimesmoresequencereadsthanthe
original454FLXtranscriptome(9,165,598versus1,000,000reads),andyieldedslightlylessthanoneand
ahalftimesmoreassembledcontigsinthepreferredTrinityassembly(161,100versus115,038contigs).
ThegreatersequencingdepthoftheIlluminatranscriptomes,informedbytheresultsoftheproteomic
analysis(seebelow), allowedustoidentifytranscriptsfor32venomproteinfamiliesexpressedin
theremipedevenomglands,23ofwhichwerenotpreviouslyreported(Figures1–4). Twelveofthese
novelputativevenomtoxinswerealsoidentifiedinthevenomproteome(Figures3and5,Table2,and
SupplementaryTableS1). Allproteinfamiliesthatwepreviouslyidentifiedinthe454datawerealso
foundintheIlluminadata,andsequencesfrombothtranscriptomesareincludedinthealignmentsin
theSupplementaryMaterial.
Severalstudieshaveusedboth454andIlluminasequencingforthetranscriptomicprofilingof
venomglands,eithertoimprovetheassemblyofIlluminadatawith454data[25],tocreateamore
deeplysequencedtranscriptome[26],ortocomparetheperformanceofthetechniquesinrecovering
venomproteinfamilies[27,28]. Weknowofonlyoneothervenomicsstudythatdirectlycompared
thesesequencingplatformstoassessthediversityofvenomproteinfamiliesintranscriptomelibraries
derivedfromthesameRNAmaterial. Barghietal.[29]foundthatalthough454sequencingwasbetter
abletoidentifylongerpeptidesequences,10ofthe30conopeptidesuperfamiliesexpressedinthe
venom glands of the cone snail Conus tribblei were only present in the Illumina data. Our results
agreewiththoseofBarghietal. thatIlluminasequencingcanprovidedeeperinsightsintovenom
glandgeneexpressionthan454sequencing. However,theresultsofthetwosequencingtechnologies
Toxins2017,9,234 4of25
differthemostfortheleastexpressedtranscripts. OnlyIlluminasequencingmanagedtopickupthe
Toxins 2017, 9, 234   4 of 25 
leastexpressedproteins,suchaslectin,kazaldomainproteins,hyaluronidase,cystatin,colipase-like
proteins,andcalycin.
Toxins 2017, 9, 234   4 of 25 
(a)  (b)
(a)  (b)
FigurFei g1u. rCe o1m. Cpoamripsaornis oofn  coof nctoingt ingu nmumbebresr sa nandd t hthee d diivveerrssiittyy  ooff  ppuutatatitvive eto txoixni nfa mfaimlieisli iedse indtiefnietdif iiend th ien  the 
Figure1.Comparisonofcontignumbersandthediversityofputativetoxinfamiliesidentifiedinthe
454 F4L5X4  FTLitXa nTiiutamni u(am)  (aan) da nIdll uIlmluimnain Ma MiSieSqe q( b(b) )v veennoomm  ggllaanndd  ttrraannscsrcirpiptotmomese osf  oXf ibXailbbaanlbuasn tuuslu tmuelunsmise nsis 
454FLXTitanium(a)andIlluminaMiSeq(b)venomglandtranscriptomesofXibalbanustulumensis
sequesneqcueden fcreodm fr tohme  tshaem saem ReN RAN sAa msamplpel.e A. Abbbbrerevviaiattiioonnss::  CCAAPP::  CCyyssteteinine‐er‐ircihc h(C (RCIRSPIS),P A),n Atignetnig 5e (nA 5g 5(A), g5), 
sequenced from the same RNA sample. Abbreviations: CAP: Cysteine-rich (CRISP), Antigen 5
and (aAPnagdt5 h),PoaagntehdnoPegsaeitnsh‐eorsgeisel‐anrteeesldaist -erd(eP lar(t‐Pe1rd)‐ 1()Pp rrp-o1rt)oeptienriosnt;se ;i nCCsS;SαCαβSβ:α:  βCC:yyCssyttisinntieen‐‐esst-tasatbabibliilizliiezzdee ddα α‐αh-h‐ehelilexilx/iβ/x‐β/sβ-hs‐ehsehete etemmt ootmtifif;o ;tIIiCCf;KK ::I CK: 
InhibIIninthohiribbyiit toCorryyys CtCiynysestt iiKnneen KoKtnn omotto mmtiooft;t iifdf;;I ddCIICKCK:K :d: dodououubbblellee I ICICCKKK  pppeeeppptttiiidddeee;;; L LLDDDLLLAAA:: l:lo olwwow dde ednnessinittysyi ltliyipp oloippprroooptteeriionnt rereiencce eprpettocorerp  tor 
class A domain; sIGFBP‐rp: single Insulin‐like Growth Factor Binding Protein‐domain related peptide; 
classc Ala sdsoAmdaoimn;a siInG;sFIBGPFB‐rPp-:r psi:nsginlge lIenIsnusulilnin‐l-ilkikee GGrroowwtthh FFaaccttoorrB BinindidnigngPr Porteoitne-idno‐mdoaimnareinla rteedlapteepdt pideep;tide; 
VEGF: Vascular Endothelial Growth Factor; Xn: xibalbins, which are new peptides identified in this 
VEGVF:E VGaFs:cVualsacru lEanrdEondthoethliealli aGlrGorwowthth FFaacctotorr;; XXnn:: xxiibbaallbbiinnss,,w whhicihcha raerne enwewpe ppteipdetisdiedse nidtiefinetdifiinedth iins this 
study (see Table 2). 
study(seeTable2).
study (see Table 2). 

Figure 2. Bar chart showing the 32 identified protein families and the diversity of contigs that passed 

the expression level threshold (see Materials and Methods) for the venom gland (red) and whole body 
transcriptomes (blue). See also Supplementary Tables S2 and S3 for further details. 
Figure 2. Bar chart showing the 32 identified protein families and the diversity of contigs that passed 
Figure2.Barchartshowingthe32identifiedproteinfamiliesandthediversityofcontigsthatpassed
the etxhpereexspsrioesns iloenvelelv tehlrtehsrheoshldol (dse(see eMMataeterriaiallss aanndd MMeetthhooddss))f oforrt htheev evneonmomgl agnldan(rde d(r)eadn)d awndho wlehbooldey body 
transtcrarinpstcorimpteosm (ebslu(bel)u. eS)e.eS eaelsaols SouSpupplpelmemeennttaarryy TTaabblleessS S22a annddS 3S3fo frofru fruthretrhdere tdaieltsa.ils.
Toxins2017,9,234 5of25
Toxins 2017, 9, 234   5 of 25 
Toxins 2017, 9, 234   5 of 25 

Figure 3. Bar chart showing the relative expression levels (FPKM values) of protein families in the 
Figure3. Barchartshowingtherelativeexpressionlevels(FPKMvalues)ofproteinfamiliesinthe
Figure 3. Bar chart showing the relative expression levels (FPKM values) of protein families in the 
venom gland transcriptome. Presence in the proteome is indicated by the P below the columns; see 
venomglandtranscriptome.PresenceintheproteomeisindicatedbythePbelowthecolumns;seealso
venom gland transcriptome. Presence in the proteome is indicated by the P below the columns; see 
also Figure 5 and Supplementary Table S1. The 10 most abundantly expressed transcripts are 
Figure5andSupplementaryTableS1.The10mostabundantlyexpressedtranscriptsarehighlighted
also Figure 5 and Supplementary Table S1. The 10 most abundantly expressed transcripts are 
highlighted by the rank numbers below the bars. For more details see Supplementary Table S2. 
bytheranknumbersbelowthebars.FormoredetailsseeSupplementaryTableS2.
highlighted by the rank numbers below the bars. For more details see Supplementary Table S2. 

Figure 4. Bar chart showing the relative expression levels (FPKM values) of protein families in the 
Figure 4. Bar chart showing the relative expression levels (FPKM values) of protein families in the 
Fwighuorlee b4o.dBya rtrcahnasrctrisphtoowmien. gThthee 10re mlaotisvte abexupnrdeasnsitolyn elxepvreelsss(eFdP tKraMnsvcarilputess a)roef hpirgohtleiginhtfeadm bilyie tshein rathnek 
whole body transcriptome. The 10 most abundantly expressed transcripts are highlighted by the rank 
wnuhmolebebrosd byeltorawn sthcrei pbtaorms. eF.oTrh meo1r0em doetsatialsb usened Saunptlpyleexmpernestasreyd Ttraabnlsec Sri3p. tsarehighlightedbytherank
numbers below the bars. For more details see Supplementary Table S3. 
numbersbelowthebars.FormoredetailsseeSupplementaryTableS3.
Toxins2017,9,234 6of25
Toxins 2017, 9, 234   6 of 25 

Figure 5. SDS‐PAGE gel of crude venom of X. tulumensis stained with Coomassie blue (left), and the 
Figure5.SDS-PAGEgelofcrudevenomofX.tulumensisstainedwithCoomassieblue(left),andthe
identified venom proteins (right). The excised bands are indicated with numbers on the right side of 
identifiedvenomproteins(right).Theexcisedbandsareindicatedwithnumbersontherightsideof
the gel. See Supplementary Table S1 for the identity of matching contigs in the transcriptome. 
thegel.SeeSupplementaryTableS1fortheidentityofmatchingcontigsinthetranscriptome.
Table 2. Peptide families identified by proteomic analyses of the venom. Canonical cysteines and 
Table2. Peptidefamiliesidentifiedbyproteomicanalysesofthevenom. Canonicalcysteinesand
sequence motifs are marked in red where known, while brackets in the cysteine pattern of the double 
sequencemotifsaremarkedinredwhereknown,whilebracketsinthecysteinepatternofthedouble
ICK domain peptide denote length of the inter‐domain linker. Predicted mature length refers to the 
ICKdomainpeptidedenotelengthoftheinter-domainlinker.Predictedmaturelengthreferstothe
predicted length of the mature peptide rather than of the contig. Contigs VG specifies the number of 
predictedlengthofthematurepeptideratherthanofthecontig.ContigsVGspecifiesthenumberof
contigs matching the mass spec fragments for each peptide. FPKM VG records the cumulative FPKM 
convtailgusems faotrc hthineg cothnetigmsa wssitshp eac mfraatgchminegn tmsfaosrs esapcehctproempteitdrey. fFrPagKmMenVt.G Sereec Sourdpspltehmeecnutmaruyl aTtaivbeleFs PSK2 M
valauneds Sf3o rfotrh aedcdointitoingasl wcointhtigasm foart tchheisneg pmepatsidsessp tehcattr olamcke tmryatfcrhaignmg menats.s SsepeecStruopmpeletrmy efrnatgamryenTtasb. lSeese S2
andSuSp3pfloemraedndtairtiyo nTaalblceo nS4ti gfosrf othret hneasmeepse opft itdhees itnhdaitvlidacukalm paetpcthidinegs, mnaamssesdp aeccctroormdinetgr ytof rtahgem raetniotsn.aSl ee
Sunpopmleemncelnattaurrye fToarb vleenSo4mf oprepthtieden taomxienss odfevtheeloipneddi vinid [3u0a,l31p]e. pAtbidbreesv,inaatimonesd: IaTcPc/oCrHdiHn:g Ioton Tthraenrsaptoiortn al
nomPeepntcidlaet/uCrreufsotarcveeanno Hmyppeerpgtliydceetromxiicn Hsdoermveolnoep;e adlli not[h3e0r, 3a1b]b.rAevbibarteiovnias taiosn ins: FITigPu/rCe H1. H1 A:I opnroTtreaonmspico rt
Pephtitid feo/r Cxribuasltbaicne a1n2 Hwyaps efrogulnycde romnliyc Hfoor ram coonnet;iga lflrootmhe trhaeb 4b5r4e vdiaattiao, nfosra swihnicFhig FuPreK1M. 1vAaluperso taeroem niocth it
foraxviabialalbbilne. 12wasfoundonlyforacontigfromthe454data,forwhichFPKMvaluesarenotavailable.
Peptide  Structural  Predicted 
PeptidFeamFaimlyi ly StrucFtuorladl Fold SSccaafffoflodld  PMredaitcutreed LMeantgutrhe  CoCnotnigtisg VsVGG  FPKFPMK MVGV G
Length
Xibalbin 1  ICK  xCx6Cx6CCx4CxCx6CxCx 49  1  7138.77 
XXXXXiiibbbiiaaabblllaabbblliiibbnnnii231nn  32   PDPDuuototauautbItbiCivlvleeKee  IIIICCCCKKKK   xxCCx6xC6CxC6CxxC6x5CxCC5CCCxxx4x5x6CC54CCCxxx9x6x–6C691CC–1x1CC1x7CC7x[C4xC[C4xCx]4xx4]CC C4CCxxx6x6xCCC6CCxxx8x6xCC8C6CxCxCCxCxCx4xCx4Cx9xC9Cxx 7744455––907079 9  21  112 1715,,1731715631,,60313..6387860.707..  78770
XXibiablablibni4n 4  PPuuttaattiivvee CCSSααββ xxCCx1x21C2Cxx3C3Cxx5C5Cxx5C5Cxx5C5CxxCCxCxC 6611  1 1 17,1077,30.7439. 49
XXibiablablibni5n 5  PPuuttaattiivvee CCSSααββ xxCCx1x41C4Cx8xC8CxCxCx3xC3Cx1x71C7CxxCCx4xC4Cx1x01C0CCCxx 8877  1 1 114101.4100. 10
XXXXXiiibbbiiaaabblllaabbblliiibbnnnii687nn  76   sIsIUITTIGGnPPFkF//CBnBCPoHPHw‐-HrrHnpp   CCxx22CCxx44CCxxxC55CCxxx1xCx08C8CCxCx1xx5x241C845CCCCxxCx2x7C72CxCCx4xCx1x9291CxCC27CxxxC53x5CxCC31Cxx0xCC8xCCx8xCx2x22C1xCCxxx22CCxx66CCxx44CCxx  17887072527   12  121 1107.194.10162.7 7.95. 6167
XXibiablablibni9n 8  UUnnkknnoowwnn  xCxxC5Cx1x0CCxx22C8CxC21xC4xC3xC7xC3xC10xC2Cx2x1>C50x  111045  1 1 4224.5,363 4.56
XiXbaiblbailnbi1n0 9  UUnnkknnoowwnn  xCx5CxCxCx2xCCxx241CCxxC3Cxx3Cx2Cx>50  12114  1 2 24,3373,45.7596. 07
Xibalbin11 Unknown Nocysteines;twoP-richdomains 18/32 2 46,289.60
XXibiablablibni1n2 10  UUnnkknnoowwnn  Nocysteines;multixpClex‘CSIxF4CQxKC’/x‘ FIFPK’domains 251  2 2 37,5790.017 
Xibalbin 11  Unknown  No cysteines; two P‐rich domains  18/32  2  46,289.60 
Xibalbin 12  Unknown  No cysteines; multiple ‘SIFQK’/‘FIFPK’ domains 5  2  0 1
Toxins2017,9,234 7of25
2.2. RevisingtheVenomicProfileof X.Tulumensis
The three most highly expressed venom protein families (fragments per kilobase million
(FPKM)>100,000)inthevenomglandaretheenzymeschitinase,peptidaseS1withLDLadomains,
and peptidase S1 without LDLa domains. The next six most highly expressed protein families
(FPKM>10,000)aresixnewpeptidefamilies(xibalbins2–4, 9–11)thatrepresentdifferentpeptide
scaffolds,includingaputativeICKpeptide,adoubleICKpeptide,apeptidewithaputativeCSαβ
fold,twocysteine-richpeptideswithunknownstructuralfolds,andtwopeptideswithoutcysteines.
Ofthe23remainingvenomproteinfamilies,nineareexpressedatFPKMvaluesbetween1000and
10,000,while14arepresentonlyatmuchlowerexpressionlevels(FPKM≤311).
Our proteomic analyses confirmed the presence in the venom of 19 of the 32 venom protein
familiesidentifiedinthetranscriptome,includingthe12mosthighlyexpressedfamilies(Figures3
and5,Table2andSupplementaryTableS1). Theonlytwoproteinfamiliesthatwereexpressedinthe
venomglandtranscriptomeatmoderatelyhighlevels(FPKMbetween1000and10,000)thatwerenot
observedinthevenomproteomearemetallopeptidaseM12andpeptidaseS10. Withtheexceptionof
xibalbin1,whichweidentifiedonthebasisoftranscriptomedatainourpreviousstudyasapeptide
withsimilaritiestoß/δagatoxins,allnovelpeptidesreportedherewereidentifiedwiththehelpofour
proteomicanalyses. Interestingly,withtheexceptionoftheverylowlyexpressedpeptidesxibalbin7
and8,allvenompeptidescouldalsobeidentifiedinthe454FLXvenomglandtranscriptomeofour
previousstudy[12].
Thegeneexpressionprofileofthewholebodytranscriptomeisstrikinglydifferentfromthatof
thevenomglands(Figures3and4). Onlytwoproteinfamiliesarerankedinthetoptenmosthighly
expressedfamiliesofbothdatasets: chitinaseandpeptidaseS1withoutLDLadomains. Eightvenom
proteinfamilies,includingfiveofthetenmosthighlyexpressedvenomproteinfamiliesinthevenom
gland (xibalbins 2–4, 10, 11), are not detected at all in the whole body transcriptome. These may
thereforerepresentvenomglandspecificproteins. Conversely,sixofthetenmosthighlyexpressed
proteinfamiliesinthewholebodytranscriptome(calycin,kazal,kunitz,lectin,phospholipaseA and
2
xibalbin8)areexpressedonlyatmoderatelytoextremelylowlevelsinthevenomglands. Interestingly,
eightofthetenvenomproteinfamiliesmosthighlyexpressedinthevenomglandsalsohavehigher
contigdiversitythaninthewholebodytranscriptome,whichsuggeststissue-specificregulationof
contigexpression(Figure2).
A recent study [32] provided a manual approach to deal with the problem that widely used
assembly software, such as Trinity, may erroneously assemble highly similar but distinct reads
into chimeric contigs, and that a failure to recognize this may underestimate the true diversity of
contigs. Oneconsequencewouldbetheoverinflationoftheexpressionlevelsofsuchchimericcontigs.
This could potentially affect our results. However, performing a manual assessment of this issue
followingthestrategyofMacranderandcolleagues[32]isunfeasiblegiventheenormousnumberof
readsineachassembly. Moreover,ourvenomglandtransciptomewasgeneratedfromthepooled
venomsystemsof25individuals. Therefore,giventhelackofadditionaldataconcerninggenecopy
numbersandintra-populationtoxingenediversity,weconsiderselectivelysplittingandreadjusting
expression levels for venom-encoding contigs to see if they might represent multi-copy genes an
inappropriatestrategy. Consequently,ourresultsprovideconservativeestimatesofthecontigdiversity
withinproteinfamilies,andexpressionlevelsofindividualcontigs,venomprotein-encodingornot,
mayinsomecasesbeoverestimates. However,whentheexpressionlevelsofallcontigswithingiven
proteinfamiliesaretakentogether,theyprovideaccurateestimatesoftherelativeexpressionlevelsof
thosefamilies(Figures3and4).
OurnewinsightsintothevenomofX.tulumensisnecessitaterevisingtheprevioushypothesisthat
remipede venom is dominated by enzymes [12]. Although chitinase and peptidase S1 enzymes
are indeed the most abundant venom components, a diversity of peptides that are highly or
uniquely expressed in the venom glands, notably xibalbins 1–4, and xibalbins 9–11, show that
remipedevenomismorecomplexthanpreviouslythought. Thepresenceofthesenewlydiscovered
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venompeptidesalongsidethehighmolecularweightproteins,morecloselyalignsremipedevenom
composition to those of the venoms of other predatory arthropods such as spiders, scorpions and
centipedes[17,18,23,24,33,34].
2.3. NewVenomicProfileProvidesInsightsintoPutativeFunctionofVenom
Inthissectionwediscussinmoredetailthenovelandthemosthighlyexpressedvenomprotein
familiesidentifiedinthisstudy,aswellastheirpossiblefunctionalsignificance. Foradiscussionofthe
othervenomproteinfamilieswereferreaderstoourpreviouswork[12].
2.3.1. Enzymes
ThemostabundantlyexpressedproteinsintheproteomeandtranscriptomearepeptidaseS1(PS1)
enzymes(Figure3,SupplementaryTableS2andFigureS3). Likewise,PS1enzymesarerepresentedby
thehighesttranscriptdiversityofallvenomproteins,aresultthatcorrespondscloselytoourinitial
study[12]. Serinepeptidasesarepresentinabroadrangeofvenoms,buttheextraordinarilyhighlevel
ofexpressioninremipedevenomglandsmostcloselyparallelstheexpressionfoundinthevenom
glandsofsomepredatorssuchasvipers,helodermatidlizards,andcephalopods[35–39]. PS1scould
playavarietyofrolesinremipedevenom. ReptilePS1s(kallikreins)oftenactonthebloodvascular
system,activatingbloodclotting,loweringbloodpressure,andincreasingvascularpermeability[38].
However,sinceremipedesarepredatorsofinvertebrateprey,particularlycrustaceans,itisunlikely
thattheirvenomhasevolvedtotargetthevertebratecirculatorysystem. Cephalopodsprovideacloser
functionalanalogueforthepossiblerolesofserinepeptidasesinremipedevenom.
Crustaceans are important prey for octopus, squid and cuttlefish, and among cephalopods
the predatory role of venom is best understood for octopuses. Octopuses drill tiny holes into the
shellsofmolluscsandtheexoskeletonofcrustaceansthroughwhichtheyinjecttheirvenom[40–42].
Thevenomparalysesandkillstheprey, butitalsofacilitatesfeedingthroughpre-digestionofthe
prey’ssofttissues. Octopusvenomproteasesspecificallytargetmuscleattachmentsitesincrabs,which
allowstheoctopustoeasilyextracttheirmuscletissue[43–45]. Inaccordancewiththisvenomrole
serineproteasetranscriptsarethemosthighlyexpressedvenomproteintranscriptsinbothoctopusand
cuttlefishposteriorsalivaryglands[35,36,46,47]. AsimilarlyhighlevelofPS1expressioninremipede
venomglandsiscompatiblewiththeideathattheytoousetheirvenomtodetachthesofttissueof
theirpreyfromtheexoskeleton,helpingwithpreyingestionandpre-digestion. However,thediversity
ofvenomPS1salsosuggestsafunctionalradiationmayhavetakenplaceandthatthisproteinfamily
mayplayadditionalrolesinremipedevenom.
The diversity of PS1s expressed in remipede venom glands includes forms with and without
lowdensitylipoproteinreceptorclassA(LDLa)domains. Interestingly,proteinswithLDLdomains
are relatively rare in venoms, and to our knowledge have only been found in arthropod venoms.
Beyondcentipedevenoms[32],whicharerichinproteinswithLDLadomains,proteinswithLDLa
domainshavebeendescribedfromthevenomoftheparasitoidwaspsNasoniavitripennis[48]and
Pteromalus puparum [49], from the skin and bristles of the caterpillar of Lonomia obliqua [50], from
the venoms of the widow spiders Latrodectus hesperus, L. geometricus and Steatoda grossa [51,52],
from the venom of the kissing bug Rhodnius neglectus [53], and from the venom of the scorpion
Hadrurusspadix[34]. Thebiologicalrolesoftheseproteinsinthesetaxaremainsunknown. However,
thestructureoftheLDLavenomproteinsinremipedesinthecontextoftheirdietprovidecluestoa
possiblerole.
The LDLa domain plays a central role in cholesterol metabolism in mammals by binding to
lipoproteins,whichfacilitatestheirtransportacrosscellmembranesviaendocytosis,andLDLdomains
ininsectsplaysimilarrolesinlipidtransport[54–56]. ThestructureoftheremipedevenomLDLa
proteins is suggestive of a role in lipid metabolism as well. Compared to the proteins with LDL
domainsinthevenomsoftheothertaxalistedabove,theremipedesequencesareuniqueincontaining
one,four,five,orsixLDLadomainsfollowedbyaPS1domain. PS1splayacrucialroleinprotein
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digestionininvertebrates[57]. Theuniquestructureoftheseremipedevenomproteinssuggeststhat
theLDLadomainsconferaffinityforlipoproteins,whilethePS1domainfacilitatestheirdigestionby
hydrolysingtheirpeptidebonds. Lipoproteinsareabundantlypresentincrustaceansandperforma
varietyoffunctions,includinglipidtransport,hemolymphclotting,defenceagainstmicroorganisms,
and lipid storage in eggs [58]. The highly expressed LDLa domain enzymes present in remipede
venommayallowthemtoeffectivelydigestandabsorbthelipoproteinspresentintheircrustacean
prey. ItmaybenoteworthythatthecavesystemwhereX.tulumensiswascollectedhousesadense
populationofatyidshrimp,Typhlatyapearsi. Althoughtheypredominantlyinhabitthebrackishwater
abovethehalocline,theydopenetratetheunderlyingmarinewater,whereremipedescanpreyupon
them. Sinceaveryhighpercentageoftheseshrimpwereobservedtobegravid[59],itmakesthema
particularlylipid-richmeal.
Chitinasesarethesecondmostabundantlyexpressedvenomprotein,aswefoundpreviously[12].
Werecovered13chitinasecontigsinourvenomglanddatathatsatisfiedtheexpressionlevelcut-off,
and 8 in the body tissue transcriptome, which suggests that chitinases exclusively expressed in
thevenomglandsmaybeundergoingadiversificationthatcouldunderpinafunctionalradiation.
Insupportofthishypothesis,theresiduescrucialforchitinaseactivitywereconservedinallseven
bodytissuecontigsthatwereofsufficientlengthtoincludethecatalyticdomain,butnotinfourof
the venom gland contigs. While three of these venom gland contigs (c30447_g1_i1; c93564_g1_i1;
c137531_g1_i1)areexpressedatsuchlowexpressionlevelsthattheyfailedourexpressionlevelcut-off,
the fourth contig (c27030_g1_i1) is the third most highly expressed chitinase contig in the venom
glandsandwasdetectedinthevenomproteome. Interestingly,thechitinaseencodedbythiscontig
hassubstitutedthecriticalcatalyticglutamicacidforglycineinalignmentposition341. Thislikely
abolisheschitinolyticactivityasglutamicacidistheprotondonornecessaryfortheenzyme’sactivity.
Butgivenitshighlevelofexpressionitmayplayanotherroleinthevenom,enzymaticorotherwise.
Our phylogenetic analysis shows that remipede chitinases group together in several distinct
parts of the chitinase tree (see Supplementary Figure S2). Interestingly, all chitinases found in the
venomglandproteomeandthetopsevenmosthighlyexpressedchitinasetranscripts(expressedone
tothreeordersofmagnitudemorehighlythantheothers)allclustertogetherinasingleclade. Thetwo
chitinasecontigsthataremosthighlyexpressedinthewholebodytranscriptomeareplacedintwo
differentcladeselsewhereinthetree.
Chitinasesareknownfromanumberofanimalvenoms,mostlyfromthevenomsofarthropod
predators such as glycerid polychaetes, cephalopods, centipedes, scorpions, spiders, cnidarians,
and hymenopterans [32,33,35,36,47,52,60–63]. But chitinase expression in remipede venom glands
is probably unparalleled. Chitinase transcript c29839_g1_i1 is the single most highly expressed
transcriptinthevenomglandsoftheremipede. Theexpressionofthisonetranscriptishigherthanthe
expressionofalltranscriptscombinedwithineachoftheothervenomproteinfamilies,exceptPS1s
withLDLadomains.
Cephalopods, especially octopuses, again provide an instructive functional analogue for
the probale role of this venom protein in remipedes. Octopuses, like remipedes, are expert
crustaceanpredators,andchitinaseisexpressedatrelativelyhighlevelsintheirposteriorsalivary
glands[35,36,43,64]. Octopuses probably do not use their venom chitinase to derive significant
nutritionalbenefitfromeatingtheirprey’schitinousexoskeleton;theyarecarefulnottoingesttoo
muchexoskeleton[64]. Thereisinsteadsuggestiveevidencethatoctopusesusetheirvenomchitinases
asacrustaceancanopener.Crustaceanmusclesinsertontheirchitinousendocuticle,andbyweakening
thisattachment,chitinasescanassistPS1sinretrievinganddigestingtheprey’smuscle[64]. Inone
publishedexperimentcrabsweretakenfromanoctopusaminuteandahalfafteritcapturedthem.
Halfanhourlaterthecrab’slegmusclesslidoutoftheirexoskeleton“likeastringofsausages”[45]
(pp. 443–444). Wesuspectthatremipedesdeploytheirvenomchitinaseinasimilarmanner. Inoneof
thefewpublishedfieldobservations[65],aremipedewasseentofeedoncrustaceanprey,afterwhich
itreleasedanemptyexoskeletalhusk. Althoughanadditionalroleofchitinasesasaspreadingfactor
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forremipedevenomispossibleaswell,wesuspectthatitsmainfunctionisdisintegratingthebodily
integrityofcrustaceanprey.
2.3.2. Non-EnzymaticProteins
The most highly expressed non-enzymatic, non-peptidic protein family in the venom of
X.tulumensisisvascularendothelialgrowthfactor(VEGF)-likeprotein. Itispresentinthevenom
proteome and is predominantly represented by a single highly expressed contig (c29360_g3_i1;
see Supplementary Table S2). VEGF-like proteins have been detected in the venoms of viperid,
elapid and colubrid snakes, as well as anguimorph lizards, the platypus, and hymenopteran
venoms [19,37–39,66–70]. VEGF is a potent inducer of vascular permeability, and can thus act as
aspreadingfactorforthevenom,aswellasassistinpreycapturebyproducingrapidhypotensionand
shock. However,VEGF-likeproteinscanbeexpressedinmanytissues,andmaybelowlyexpressedin
venom,suchasincolubridsnakes,suggestinginsteadanendophysiologicalrole[66]. Therelatively
highexpressionlevelofVEGF-likeproteinintheremipedevenomglands,comparedtoitslowlevel
ofexpressioninthewholebodytranscriptome,suggeststhatitmayplayaroleinremipedevenom.
Sinceremipedeshavenotbeenseentopreyonvertebrates(althoughapossiblesporadicinfluxof
larvalopenwaterfishintotheircavesystem[59]doesnotprecludethepossibilitythatremipedes
couldoccasionallypreyontinyfish),thisvenomproteinprobablydoesn’tplayaroleinpredation.
Ithasbeenproposedthatinhymenopteranswithcytolyticvenoms,VEGFmayhelpmaintainanintact
venomglandbypromotingcellgrowth[19,70]. Althoughasimilarroleisconceivableinremipedes
(xibalbin10isapossiblecandidatecytolytictoxin;seeSection2.3.3),VEGFmightalsoplayarolein
thedefensiveuseofvenom. Whenmanipulatingcollectedremipedeswithforceps,theyattack,grab
andaudiblybiteitwiththeirvenomdeliveringmaxillules. Itispossiblethatwhentakeninthemouth
bycavefish,X.tulumensismaydeliveradefensivebite,withVEGF-likeproteintargetingthefish’s
circulatorysystemtocausearapiddropinbloodpressure. Theeffectivenessofhypotensivevenom
peptidesinfacilitingescapefromfishpredatorshasrecentlybeendemonstratedforfangblennies[71],
andremipedesmightrelyonasimilarstrategy.
2.3.3. Peptides
Contrarytotheprevioustranscriptome-basedperceptionofremipedevenomasbeingcomposed
almostexclusivelyofenzymes,ourrevisedvenomprofileofX.tulumensisshowsthatitalsocontains
several novel putative neurotoxin-like peptides. Although the contig diversity is low compared
to other predatory arthropod venoms, the peptides we identified in the venom of X. tulumensis
compriseasignificant13familiesspanningatleastnineuniquestructuralscaffolds,fourofwhichlack
significantsimilaritytoanyknownstructuralclass.Thesepeptidesalsoincludesomeofthemosthighly
expressedvenomcomponentsidentified—thetranscriptencodingtheputativeICKU-xibalbin2-Xtu1a
is the second highest expressed contig in the venom gland transcriptome (c29772_g1_i1, FPKM
75,130.8),secondonlytoapresumablyenzymaticallyfunctionalchitinaseisoform(c29839_g1_i1,FPKM
178,137.36). Peptides,andespeciallycysteine-richpeptides,arethereforelikelytoplayagreaterrolein
thevenomofremipedesthanpreviouslythought,mostlikelyasprimarilyneurotoxiccomponents.
ICK peptides are among the most abundant cysteine-rich peptides known, and are found in
anexceptionallywiderangeoforganismswheretheymostlikelyactasdefensemoleculesagainst
pathogens (“defensins”) [72]. ICK peptides have also been identified in venoms from a number
ofanimallineages, andconfirmingtheirpresenceinthevenomofX.tulumensisthereforecameas
no surprise. However, in addition to the previously reported xibalbin 1 and the extremely highly
expressedxibalbin2,wealsoidentifiedahighlyunusualfamilyofdoubleICKdomain(dICK)peptides,
xibalbin3. InstarkcontrasttotheubiquityofsingleICKdomainpeptides,dICKshavepreviouslyonly
beendescribedfromvenomsfromthemygalomorphspidersHaplopelmaschmidtii(tau-TRTX-Hs1a,
henceforth Hs1a) [73] and Hadronyche infensa (pi-HXTX-Hi1a, henceforth Hi1a) [74] as well as the
araneomorphspiderCheiracanthiumpunctorium(delta-MGTX-Cp1,henceforthCp1)[75]. Inthecases