0.0255556.com

BioMedCentralPage1of9(pagenumbernotforcitationpurposes)BMCEvolutionaryBiologyOpenAccessResearcharticleExpressionpatternofthree-fingertoxinandphospholipaseA2genesinthevenomglandsoftwoseasnakes,LapemiscurtusandAcalyptophisperonii:comparisonofevolutionofthesetoxinsinlandsnakes,seakraitsandseasnakesSusantaPahari1,4,DavidBickford1,BryanGFry1,2andRManjunathaKini*1,3Address:1ProteinScienceandConservationEcologyLaboratories,DepartmentofBiologicalSciences,NationalUniversityofSingapore,117543,Singapore,2DepartmentofBiochemistry&MolecularBiology,Bio21Institute,UniversityofMelbourne,Parkville,Victoria,3010Australia,3DeparmentofBiochemistry,MedicalcollegeofVirginia,VirginiaCommonwealthUniversity,Richmond,VA23298-0614USAand4CenterforPostGraduateStudies,SriBhagawanMahaveerJainCollege,18/3,9thMain,Jayanagar3rdBlock,Bangalore,IndiaEmail:SusantaPahari-susanta2001@yahoo.
com;DavidBickford-dbsbdp@nus.
edu.
sg;BryanGFry-bgf@unimelb.
edu.
au;RManjunathaKini*-dbskinim@nus.
edu.
sg*CorrespondingauthorAbstractBackground:Snakevenomcompositionvarieswidelybothamongcloselyrelatedspeciesandwithinthesamespecies,basedonecologicalvariables.
Interrestrialsnakes,suchvariationhasbeenproposedtobeduetosnakes'diet.
Landsnakestargetvariouspreyspeciesincludinginsects(arthropods),lizards(reptiles),frogsandtoads(amphibians),birds(aves),androdents(mammals),whereasseasnakestargetasinglevertebrateclass(fishes)andoftenspecializeonspecifictypesoffish.
Itisthereforeinterestingtoexaminetheevolutionoftoxinsinseasnakevenomscomparedtothatoflandsnakes.
Results:Herewedescribetheexpressionoftoxingenesinthevenomglandsoftwoseasnakes,Lapemiscurtus(Spine-belliedSeaSnake)andAcalyptophisperonii(HornedSeaSnake),twomembersofalargeadaptiveradiationwhichoccupyverydifferentecologicalniches.
WeconstructedcDNAlibrariesfromtheirvenomglandsandsequenced214and192clones,respectively.
Ourdatashowthatdespitetheirexplosiveevolutionaryradiation,thereisverylittlevariabilityinthethree-fingertoxin(3FTx)aswellasthephospholipaseA2(PLA2)enzymes,thetwomainconstituentsofLapemiscurtusandAcalyptophisperoniivenom.
Tounderstandtheevolutionarytrendsamonglandsnakes,seasnakesandseakraits,pairwisegeneticdistances(intraspecificandinterspecific)of3FTxandPLA2sequenceswerecalculated.
Resultsshowthattheseproteinsappeartobehighlyconservedinseasnakesincontrasttolandsnakesorseakraits,despitetheirextremelydivergentandadaptiveecologicalradiation.
Conclusion:Basedontheseresults,wesuggestthatstreamlininginhabitatanddietinseasnakeshaspossiblykepttheirtoxingenesconserved,suggestingtheideathatpreycompositionanddietbreadthmaycontributetothediversityandevolutionofvenomcomponents.
Published:27September2007BMCEvolutionaryBiology2007,7:175doi:10.
1186/1471-2148-7-175Received:20April2007Accepted:27September2007Thisarticleisavailablefrom:http://www.
biomedcentral.
com/1471-2148/7/1752007Paharietal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
BMCEvolutionaryBiology2007,7:175http://www.
biomedcentral.
com/1471-2148/7/175Page2of9(pagenumbernotforcitationpurposes)BackgroundThecompositionofsnakevenomsvarieswidelybothwithinaspeciesandamongcloselyrelatedspecies[1-4].
Thisvariationisproposedtobeduetochangesinthedietofsnakes,basedonthefindingsinthevariationofintraspecificvenomcompositioninapitviper,Callose-lasmarhodostoma,alandsnake[2].
Landsnakesdependonadiversityofpreyincludinglizards(reptiles),frogsandtoads(amphibians),birds(aves),insects(arthropods),androdents(mammals)[5,6].
Theyprobablyrequirearangeoftoxinsthattargetdifferentgroupsofpreyspeciessincethereisvariationinvenom'sabilityforimmobiliza-tionandkillingacrosssuchavarietyofprey.
Toxinswhichareusedforsystematicpreyenvenomationfoundtohaveseveralisoformsintheirvenomglandasevidentfromglo-balcataloguingofsnakestoxingeneexpression[3,7-16]andithasbeencorrelatedthatvariationinpreyfavorstheevolutionofmultipleisoformsoftoxinsinvenoms[9,17].
Thevarietyofisoformsisbelievedtohavebeenachievedthroughfrequentgeneduplicationsaccompa-niedbyanacceleratedrateofevolution[18-20]similartothegenerationofadaptiveresponseinimmunoglobulinsandmajorhistocompatibilitycomplexgenesinresponsetoawiderangeofforeignantigens[21].
Thus,abirth-and-deathmodeofevolutiongeneratesdiversityintoxinsallowingsnakestofeedonavarietyofpreyspecies[22].
Elapidsnakesareamonophyleticcladeofapproximately300speciesin61genera[23].
Trueseasnakes(Hydrophi-inae)andseakraits(Laticaudaspp.
)formtwoelapidcladesthathaveevolvedindependentlybutareeitherrootedwithin(trueseasnakes)orbasalto(seakraits)theterrestrialAustralo-Papuanelapidsratherthanotherelapidgroups[24-28].
Thesesnakeshaveadaptedtomarinelifeandundergonemanychangesinforagingbehavior,morphologyanddiet[29].
Asaresult,althoughtheirfeedingsystemsareconfinedtopreyofasinglever-tebrateclass(fishes),theyoftenspecializeonparticulartypesorfamiliesoffish[30,31].
Withsuchrestrictionsinbothdietandhabitat,onemightexpectlowdiversityintoxincomponents(relativetosnakeswithbroaderdiets),ashasbeenshowntobethecaseinthehydrophiinaesub-family[9].
WeshowedbyanalyzingthecDNAlibraryofAipysuruseydouxiithatits3FTxgeneisinactivatedbyadinucleotide(TT)deletion[32]andtheevolutionofitsPLA2isoenzymes,unlikethosefromothersnakevenoms,isdecelerated[33].
Asthisuniqueseasnakefeedsexclu-sivelyfisheggs[31],wesuggestedthatashiftinthedietofA.
eydouxiimayhaveresultedintherelaxationofselectionpressuresonits3FTxandPLA2genesHere,weexaminedthetotalgeneexpressionpatternoftwootherseasnakes,LapemiscurtusandAcalyptophispero-nii,whichhavedistinctanddifferenthabitatsandfeedingsystems.
L.
curtusinhabitsmanydifferentareaslikeopensea,estuaries,andcoralreefs,whereasA.
peroniiinhabitsonlysandyareasbetweencoralreefs[34].
L.
curtusincon-trasttootherseasnakesisageneralistfeederanditsdietisoneofthemostdiverseofallseasnakes[30,31,34-36].
Itspreyconsistsoffishes(90%;31differentfamilies)andveryfewinvertebrates(10%;squidandcuttlefish)[30,35,36].
Additionally,L.
curtuscohabitswithotherseasnakes,andconsequentlymaybeoverlappingindiet.
Incontrast,thedietofA.
peroniiisconfinedmainlytogobies(oneclassofseafish)[34]anditisadietandhabitatspe-cialist.
BecausethesetwosnakesaremembersofalargeadaptiveradiationoftheHydrophislineageandtheymighthavedivergedveryrapidly,differencesintheirvenomsmightalsobewidelydivergentiftheytrackdietspeciali-zation.
Ontheotherhand,ifdietspecializationwithinaconstrainedgroupofprey(e.
g.
,onlyfish),drivesmoreofastreamliningofvenomevolution,thenwemightexpecttheretobefewornochangesinvenomconstituents.
Therefore,itcouldbeinterestingtocomparethetotaltoxingeneexpressionofthesetwoseasnakes.
WeconstructedcDNAlibrariesofthevenomglandsfromA.
peroniiandL.
curtusspecimensandsequencedabout200clonesofeach.
Samplingoftranscriptomsindicatesthepresenceofanynewand/orrarefamiliesoftoxinsandenablesanalysesofthemolecularevolutionarytrendsamongtoxingenes.
Further,tocomparetheevolutionoftoxingenesamonglandsnakes,seasnakes,andseakraits,wecalculatedtheevolutionarydistancesusingallavaila-blesequencesoftwoprinciplecomponentsofthetoxinproteome,3FTxandPLA2.
ResultscDNAlibrariesofLapemiscurtusandAcalyptophisperoniivenomglandsWeobtained4and5gofmRNAfrom30mgofvenomglandtissuesofLapemiscurtusandAcalyptophisperonii,respectively.
WeconstructedtwoseparatecDNAlibrariesusing1gofmRNAfromeachpreparation.
Fromtheclonescontaininginserts,werandomlyselected250and225clones,respectively.
Fromthesecloneswewereabletoobtainsequencesof214cDNAclonesfromL.
curtusand192cDNAclonesforA.
peronii.
Figure1showsthedistributionofclonesinbothvenomglands.
Lapemiscurtuslibrary3FTxTodate,threelong-chainisoformsof3FTx(AAL54893,AAL54892andABN54806)andfourshort-chainisoformsof3FTx(AAL54894,AAL54895,P68416andABN54805)[37]havebeenreportedfromL.
curtusvenom.
WefoundcDNAclonesencodingbothlong-chainisoforms(AAL54893andAAL54892)of3FTxinthelibrary(41%abundance,Figure1)andtheratiobetweenthenumberofclonesofisoformsAAL54893andAAL54892was~10:1.
BMCEvolutionaryBiology2007,7:175http://www.
biomedcentral.
com/1471-2148/7/175Page3of9(pagenumbernotforcitationpurposes)WealsofoundcDNAclonesencodingashort-chain3FTx(AAL54894;~2%abundance,Figure1)[37].
Novariationwasobservedinthecodingsequenceofthematurepro-teinswithAAL54893,AAL54892andAAL54894.
How-ever,wecouldnotdetectthelongchainisoform(ABN54806)andthreeothershort-chainisoformsof3FTx(AAL54895,P68416andABN54805)[37,38].
PLA2Sofar,threeisoformsofPLA2ofL.
curtus(AAL55556,AAL55555andAAL54920)havebeenreported[39].
WewereonlyabletodetectoneisoformwhichiscompletelyidenticalatthenucleotidelevelwithAAL55555(~10%abundance,Figure1).
CRISPPartialsequencesoftwoisoforms(Q8UW25andQ8UW11)ofCRISPfromL.
curtusvenomglandshaverecentlybeenreported.
OurcDNAlibrarycontained~2%clonescodingforQ8UW25isoformwithoutanyvariationatnucleotidelevel(Figure1).
OthersThecDNAlibraryhasasingletonpresenceofagrowthfac-tor(AY742212)whichshowssignificantidentitytoPlate-letDerivedGrowthFactor(PDGF).
Thepartialsequenceshows70%identitytotheC-terminusofthepredictedPDGF-DisoformfromGallusgallus(chicken).
AlthoughgrowthfactorssuchasNGF[40]andVEGF[41]areknowntobepresentinthevenom,thisisthefirstreportofPDGF-likeproteinsequencefromthevenomgland.
However,furtherstudiesareneededtoconfirmthepresenceofPDGFproteininthevenom.
L.
curtuslibrarycontained~20%housekeepinggenes(Fig-ure1),includingribosomalRNA,ribosomalproteinsandcytochromes.
Inaddition,~25%ofcDNAsequencesdidnotshowsignificantidentitytotoxinsormetabolicgenes(Figure1).
BlastXsearchofthesesequencesshowedpoororonlypartialidentitytoanyproteinsequenceswithotherorganismsornomatchatall.
Theseunknownsequencesinmostofthecasesarepartial,singletonclones.
However,theirorigin(venomglandormarginalcontaminationofsurroundingtissues)stillneedstobeestablished.
Acalyptophisperoniilibrary3FTxAminoacidsequencesoftwoisoformsofshort-chain3FTxhavebeenreportedearlier[42,43].
Gln43ofthemajorisoform(AY742211)haschangedtoGlu43intheminorisoform(AY742210)[43].
InAcalyptophisperoniilibrary,theshort-chain3FTxwasmostabundant(~64%)(Figure1)andtherearetwoisoformsof3FTxinequalnumbers(60and62respectively).
Thesetwoisoforms(AY742210andAY742211)havethreenucleotidechangesintheirsignalsequencesleadingtosubstitutionofThr7(ACC)andLeu8(TTG)bySer7(TCC)andPro8(CCG),respectively.
However,novariationwasobservedinthecodingsequenceofthematureproteinandthededucedproteinsequencecorrespondsonlytothemajorisoform[42].
AswedidnotobtainclonescorrespondingDistributionoftranscriptsinthevenomglandsofLapemiscurtusandAcalyptophisperoniiFigure1DistributionoftranscriptsinthevenomglandsofLapemiscurtusandAcalyptophisperonii.
HypotheticalsequencesHypotheticalsequencesLong-chainneurotoxinHousekeepinggenesHousekeepinggenesShort-chainneurotoxinPLA2GrowthfactorShort-chainneurotoxinPLA2CRISPLapemiscurtusAcalyptophisperoniiBMCEvolutionaryBiology2007,7:175http://www.
biomedcentral.
com/1471-2148/7/175Page4of9(pagenumbernotforcitationpurposes)totheminorform,weproposethattheminorformismostlikelyduetodeamidationofGln43[44]andnotaseparategeneproduct.
Generallyintoxinfamilies,ithasbeenobservedthatthesignalpeptideregions,5'UTRand3'UTRarehighlyconserved,whereasthematureproteinregionshowsanumberofsubstitutions[19,45].
Incon-trast,thetwoisoformsofshort-chain3FTxdifferintheirsignalpeptideregionbutnotinthematureproteininthiscase.
Itwouldbeinterestingtoexaminetheimportanceofthesesubstitutions.
PLA2SofarnoPLA2sequencesfromA.
peroniihavebeenreported.
Wefoundpartialcloneshaving3'terminalsequencesofPLA2inA.
peroniilibrary(~5%;Figure1).
Theyshow100%identityinthe3'UTRregionwithL.
cur-tusPLA2(AAL55556andAAL54920).
Furtheridentifica-tionandcharacterizationoffulllengthPLA2isunderway.
OthersThecDNAlibrarycontains~6%clonesencodinghouse-keepinggenes(Figure1).
TheseincludeNADHdehydro-genase,ribosomalproteinsandCa2+bindingproteins(calglandulin).
Thelatterclassofproteinhasbeenimpli-catedintoxinsecretion[46,47].
LiketheL.
curtuslibrary,theA.
peroniilibraryalsocontained~25%withnohomol-ogytoanyknowntoxinorhousekeepinggenes(Figure1).
Asearlier,inmostcasesthesesequencesarepartial,single-tonclonesandtheiroriginneedstobeverified.
Intraandinterspecificrelationshipof3FTxandPLA2sequencesThenumberofavailableproteinsequencesencoding3FTxandPLA2werehigherthancDNAsequencesbecausemostofthesequenceshavebeenreportedfromdirectproteinsequencing.
Therefore,weusedproteinsequencestocal-culateintraandinterspecificpairwisedistancesforlandsnakes,seasnakesandseakraits.
Itshouldbenotedthatduetopaucityoftheavailabledatathenumberofspeciesandnumberofshort-chain3FTxusedforthecalculationsforlandsnakes,seasnakesandseakraitswerenotthesame.
Forshort-chain3FTx,37%oftheintraspecificdistancesofbothPseudonajatextilisandBungarusspecies(landsnakes)areintherangeof(0.
2–0.
3)and(0.
7–0.
8)respectively,while63%oftheintraspecificdistancesofseakraitsfallintherangeof(0.
1–0.
2),andmostoftheintraspecificpair-wisedistancesofseasnakesareintherangeof(0.
02–0.
04)(Figure2A).
Interspecificpairwisedistancesalsoappearhigher(50%intherangeof0.
7forBungarusspe-cies)forlandsnakes,andlowerforseasnakes(100%intherangeof0.
02).
Interspecificdistancesof3FTxforPseu-donajaspecieswerenotcalculatedbecausesequenceswereonlyavailablefromonespecies(P.
textilis).
Thehighergeneticdistancesof3FTxinlandsnakesindicatehigherlevelsofgeneticdiversitycomparedtoseasnakes,wheresequencesweremuchmoreconserved.
Thegeneticdiver-sitywithinseakraitsisintermediateinbothintraandinterspecificcomparisons.
ForPLA2,22%oftheAustral-ianelapidsand36%oftheBungarusspeciesintraspecificdistancesfallbetween(0.
1–0.
3)and(0.
1–0.
2)respec-tively.
Ontheotherhand,97%and44%oftheseasnakes'andseakraits'intraspecificdistancesrangedfrom(0.
1–0.
2)and(0.
2–0.
3)respectively(Figure2B).
InterspecificdistancesofPLA2forAustralianelapids,Bungarusspeciesandseakraitsandseasnakeshavecomparablevalues(30%–60%intherangeof0.
2–0.
3;Figure2B).
Butinseasnakes,interspecificdistances(58%fallbetween0.
2–0.
3)appearlowerthantheintraspecificdistances.
Oneofthepossibilitiesforthisreversetrendcanbeduetopoorphy-logeneticresolutionamongspeciesinthehydrophiinaesubfamily[48,49]DiscussionSnakevenomsarearichanddiversesourceofpharmaco-logicallyactiveproteinsandpeptidecomponents[50,51].
Someofthesecomponentsareenzymes,whereasothersarenonenzymaticproteinsorpolypeptides.
Mostofthesecomponentsareoffensiveweaponstocapturetheprey,injectionofvenomintopreyleadstoimmobilization,deathandcansubsequentlyaidindigestionaswell[52,53].
Venommightalsobeusedfordefensivepurposestokeeppossiblepredatorsaway.
Venomsystemsappeartohaveevolvedtomeetsomeofthesegoals,asingletimeinreptileevolution,atthebaseoftheToxicofera[54,55].
Inthiswork,weshowthehighabundanceof3FTxinthevenomsofseasnakes(41%forLapemiscurtusand~64%forAcalyptophisperonii)whilePLA2isadistantsecondlarg-estgroup(~10%forL.
curtusand~5%forA.
peronii)ofseasnaketoxins.
Overall,boththe3FTxandPLA2donotpos-sessanabundanceofdifferentisoformsgeneratingsignif-icantvariationinthevenomcomposition.
ThefactthatwedidnotdetectsomeoftheisoformsofthesetwogroupsoftoxinsaspreviouslyreportedinL.
curtusmaybeeitherduetoregionalvariationwithinthespeciesorasamplingartifactsincethecDNAlibrarywasgeneratedfromvenomglandsofasinglesnake.
However,bothgroupsoftoxinsappearstobesimpleanddonothavenoteworthydiversityintheirisoformcompositions.
Itsuggeststhatseasnakevenomsgenesarequiteconserved,andthereforelackthediversityinitsvenomcompositionasobservedforlandsnakeandseakraits.
However,addi-tionaldatafromgeneexpressionprofile,frequencyofgeneduplicationandacceleratedevolutionprofileofseasnakesisneededtofurthertestthishypothesis.
Comparisonofintraspecificdistancesamong3FTxshowedthatthemaximumvalueforlandsnakesis0.
8BMCEvolutionaryBiology2007,7:175http://www.
biomedcentral.
com/1471-2148/7/175Page5of9(pagenumbernotforcitationpurposes)Pairwiseintraspecific(whitebar)andinterspecific(blackbar)distancesforlandsnakes,seasnakesandseakraitsFigure2Pairwiseintraspecific(whitebar)andinterspecific(blackbar)distancesforlandsnakes,seasnakesandseakraits.
PanelA:3FTx(1aand1b:landsnakes;PseudonajatextilisandBungarusspeciesrespectively),2and3:seakraitsandseasnakesrespectively.
PanelB:PLA2:(4aand4b:landsnakes;AustralianelapidsandBungarusspeciesrespectively),5and6:seakraitsandseasnakesrespectively.
RFdenotesrelativefrequency.
0%50%100%00.
020.
0400.
10.
20%50%100%20%50%100%00.
50.
1a1b00.
150.
33PairwisedistanceA100%50%0%RelativefrequencyPairwisedistance0%50%00%00.
20.
451RelativefrequencyB10%50%100%00.
20.
40.
60.
60%50%00%00.
20.
44a4b0%50%100%00.
20.
46BMCEvolutionaryBiology2007,7:175http://www.
biomedcentral.
com/1471-2148/7/175Page6of9(pagenumbernotforcitationpurposes)whereasseasnakesareat0.
03andseakraits,0.
2(Figure2A).
Thevariationbetweenlandandseasnakesisabout30fold,whereaslandsnakeandseakraitdifferonly4fold.
However,thislevelofvariationhasnotbeenfoundinPLA2genes.
Inlandsnakes,themaximumintraspecificdistanceis0.
2forlandsnakesandseakraits,whereasseasnakeshaveamaximumvalueof0.
1,indicatingadiffer-enceofonly2fold(Figure2B).
Interspecificdistances,forboth3FTxandPLA2,ontheotherhand,showgreaterorequalvaluesthantheintraspecificdifferencesinlandsnakesandseakraits(Figure2Aand2B).
Fromthegeneticdistancedata,itisobviousthat3FTxisgainingmorevari-abilitythanPLA2.
Thisisprobablyrelevantbecauseenvenomationbyelapidsnakesisusuallycharacterizedbyrapidneurotoxiccomplicationsduetopresenceoflargeamountsofneurotoxins[56].
Overall,ourcalcula-tionfortheintraandinterspecificvariationinboth3FTxandPLA2appearsdistinctamonglandsnakes,seasnakesandseakraitsindicatingtheprobableexistenceofdistinctevolutionarypatternsthatseparatethesegroups.
Interestingly,theconservationoftoxindiversityinseasnakesisnotconfinedwithinspecies,itextendsacrossdif-ferentgenera.
Forexample,Enhydrinaschistosa,acommonseasnake,hasjusttwoneurotoxins(P25492andP25493)[57].
ThetoxinP25492isidenticalinsequencetoashort-chainneurotoxinfoundinLapemiscurtusvenom[38]andtheothertoxin,P25493,isidenticaltotheshort-chainneurotoxinsfoundinvenomsofHydrophiscyanocinctus[58]andPelamisplaturus[59].
Incontrast,among2763FTxsreportedtodate[22],wecouldnotfindasingle3FTxcommonacrossdifferentgeneraoflandsnakes.
Conservationoftoxinsequences,evenacrossgeneraofmarinesnakesispossiblyduetoahighlyconstrainedniche,andthestreamlinednatureoftheirvenomsisresponsiblefortheremarkabledegreeofantivenomcross-reactivity[60].
TheanalysisofourcDNAlibrariesindicatedthattheLapemiscurtusvenomismarginallymorediversethanthatofAcalyptophisperonii.
TheL.
curtuslibrarycontainsCRISPandgrowthfactorisoformsinadditionto3Ftxneurotox-insandPLA2enzymes.
Chenetal.
(AAV98367)reportedthepresenceofakallikreintoxininLapemiscurtusvenomaswell.
RecruitmentofadditionaltoxinfamilieslikeCRISP,growthfactor,kallikreintoxinmaybeduetoitsbroaddietaryrequirements.
Incontrast,A.
peroniivenomglandscontainonlyneurotoxinsandPLA2inhighconcen-trationandittargetsonlygobiesasitsdiet[30,31,34-36].
Therefore,evolutionoftoxin(s)inageneralist(L.
curtus)andarestrictedfeeder(A.
peronii)appeartobedifferent.
Thisdoesnotindicatethatothertoxinclassesarenotexpressedatlowlevels;morerigoroussequencingmayrevealrarertranscripts.
ThetoxinexpressionprofiledatafromcDNAlibraryofL.
curtusandA.
peroniiandarelationshipbetweentheirhab-itatanddietmaysuggestthatecologicalvariablespresum-ablyplayedamajorroleindeterminingthetrajectoryoftheirevolutionarypathsalongecologicalniches(special-istandgeneralist)andnotcompletelybecauseofadistantphylogeneticrelationshipbetweenthem.
Therearehow-ever,afewspecificcasesavailableintheliterature,wherearelationshipbetweenintraspecificvariationsinvenomwithrespecttodietarypreferenceshasnotbeenfound[61,62,63].
Dothesespecificexceptionsprovethegen-eralrule,oristhereathresholdwheretheevolutionoftoxinsbecomesdecoupledfromfeedingecologyand/ordietThesequestionsremaincogentforthefutureoftoxinevolutionresearchandweproposethatseasnakeswillremainmajorplayersinhelpingtounderstandhowtoxinevolutionandfeedingecologyarelinked.
ConclusionGlobalcataloguingoftoxinexpressionshowsconservedexpressionpatternoftwomainfamiliesoftoxins,3FTxandPLA2,intwoseasnakesvenomgivingrisetoasimplevenomcompositionrelativetolandsnakesandseakraits.
Geneticdistancevaluesof3FTxandPLA2toxinsshowamorediversetrendofevolutionforlandsnakesandseakraitsthantoseasnakes.
Asthedietbreadth(preyitems)expandsfromseasnakestolandsnakes(seakraitsasinter-mediate),wesuggestthatthesetrendsinevolutionoftox-insmaybelinkedtotheirdiet.
MethodsCollectionofvenomglandsLapemishardwickiihasbeensynonymizedwithLapemiscurtus[64]soL.
curtusisusedinthispaper.
OnespecimenofL.
curtusandanotherofA.
peroniiwerecollectedfromAlbatrossBayinWeipa,Queensland,Australia.
Venomglandsweredissectedfromeachofthesefreshlycaughtsnakes.
TwoglandsfromeachsnakewereusedfortheconstructionofcDNAlibraries.
Althoughsamplesizesaresmallforeachspecies,thedifficultyinacquiringspeci-mensorkeepingindividualsincaptivitymakeeventhesesmallsamplesizesextremelyvaluableandworthyofstudy.
Libraryconstruction,sequencingandanalysisTotalRNAwasextractedfromthevenomglandsusingRNeasyMiniKit(Qiagen,Hilden,Germany).
Theinteg-rityoftotalRNAwascheckedbyagarosegelelectrophore-sis.
ThemRNAwaspurifiedusingmRNAisolationkit(RocheAppliedScience,Mannheim,Germany).
Thepuri-fiedtotalmRNAwasusedtomakethecDNAlibraryfol-lowingtheinstructionsoftheSMARTcDNAlibraryconstructionkit(Clontech,Mountainview,California,USA).
ThelibrarywaspackagedusingGigapackgoldpack-agingextract(Stratagene,CedarCreek,Texas,USA).
Indi-BMCEvolutionaryBiology2007,7:175http://www.
biomedcentral.
com/1471-2148/7/175Page7of9(pagenumbernotforcitationpurposes)vidualcloneswererescuedfromrandomlyselectedwhiteplaquesandgrownin(Luriabroth+ampicillin)medium.
PlasmidswerepurifiedusingQIAprepspinminiprepkit(Qiagen,Hilden,Germany).
PurifiedplasmidsweresequencedbycyclesequencingreactionusingtheBigDyeTerminatorv3.
1kit(AppliedBiosystem,FosterCity,Cali-fornia,USA)andwithanautomatedDNAsequencer(Model3100A,AppliedBiosystem,FosterCity,Califor-nia,USA).
SequenceswerecomparedtocDNAandpro-teinsequencesinNCBIdatabaseusingBLASTprogram(BlastNandBlastX)andidentical(orsimilar)cloneswereclustered.
EachclusterwasalignedusingtheprogramClustalWinEuropeanBioinformaticsInstitutesite.
CalculationofgeneticdistancesGeneticdistanceswerecomparedbycalculatingintraandinterspecificpairwisedistancesforthe3FTxandthePLA2enzymes.
Allavailableproteinsequencesof3FTx(short-chainisoforms)andPLA2oflandsnakes,seakraitsandseasnakeswereretrieved[seeadditionalfile1].
Redundantsequencesandsignalpeptideswereremovedandaligned.
AlignedsequenceswereanalyzedinPAUP*version4.
0program[65]usingthepairwisedistancealgorithm(uncorrecteddistances,kimura-2parameters)forbothwithinandbetweenspecies.
Thepairwisedistanceswerethenplottedasagroupforlandsnakes,seasnakesandseakraits.
AccessionnumbersNucleotidesequencedatareportedherehavebeendepos-itedinGenBankunderaccessionnumbers[GenBank:AY742212,GenBank:AY742210,GenBank:AY742211].
CompetinginterestsTheauthor(s)declaresthattherearenocompetinginter-ests.
Authors'contributionsSPhasperformedtheexperiments,dataanalysis,writingandextensionofthethemeofthemanuscript.
DBhashelpedtoexaminethephylogeneticaspectoftheconcept.
BGFisresponsibleforthesamplecollectionandwritingofthemanuscript.
RMKcontributedthedevelopingtheconceptandwritingofthemanuscript.
Alltheauthorscontributedtoeditingthemanuscriptandapprovedofitsfinalform.
AdditionalmaterialAcknowledgementsThisworkwassupportedfromthegrantsfromBiomedicalResearchCoun-cil,AgencyforScienceandTechnologyResearch,Singapore(RMK)andtheAustralianGeographicSociety,Australia&PacificScienceFoundation,Aus-tralianResearchCouncil,CASSFoundation(BGF).
).
WeacknowledgethesuggestionandhelpofDr.
RudolphMeier,Mr.
ShiyangKwongforthedataanalysis.
WewouldalsoliketothankDaveDonaldforhisinvaluablehelpincollectingspecimensinWeipa.
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