Caenorhabditis elegans as a model to screen plant extracts and compounds as natural anthelmintics for veterinary use

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j o ur na l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Caenorhabditis

elegans

as

a

model

to

screen

plant

extracts

and

compounds

as

natural

anthelmintics

for

veterinary

use

Luciana

M.

Katiki

_,

_Jorge

_F.S.

_Ferreira

,

Anne

M.

Zajac

,

Carol

Masler

,

David

S.

Lindsay

,

Ana

Carolina

S.

Chagas

_,

_Alessandro

_F.T.

_Amarante

a_{InstitutodeZootecnia(SAA,APTA),RuaHeitorPenteado56,NovaOdessa,SP,cep13460-000,Brazil}

b_{AppalachianFarmingSystemsResearchCenter(USDA-ARS),1224AirportRd.,Beaver,WV25813,USA}

c_{Virginia-MarylandRegionalCollegeofVeterinaryMedicine,VirginiaTech,Blacksburg,VA24060,USA}

d_{NematologyLaboratory,USDA-ARS,BARC-West,Bldg.011A,Rm.165B,Beltsville,MD20705-2350,USA}

e_{EmbrapaPecuáriaSudeste,Rod.WashingtonLuizkm234,SãoCarlos,SP,Brazil}

f_{UNESP-UnivEstadualPaulista,DepartamentodeParasitologia-IB,Botucatu,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13January2011

Receivedinrevisedform13May2011 Accepted16May2011

Keywords:

C.elegans

Anthelminticplants Gastrointestinalnematodes Plantextracts

a

b

s

t

r

a

c

t

Themostchallengingobstaclestotestingproductsfortheiranthelminticactivityare:(1) establishingasuitablenematodeinvitroassaythatcanevaluatepotentialproductuse againstaparasiticnematodeofinterestand(2)preparationofextractsthatcanbe redis-solvedinsolventsthataremiscibleinthetestmediumandareatconcentrationswell toleratedbythenematodesystemusedforscreening.Theuseofparasiticnematodesas ascreeningsystemishinderedbythedifficultyofkeepingthemaliveforlongperiods outsidetheirhostandbytheneedtokeepinfectedanimalsassourcesofeggsoradults whenneeded.Thismethodusesthefree-livingsoilnematodeCaenorhabditiselegansasa systemtoscreenproductsfortheirpotentialanthelminticeffectagainstsmallruminant gastrointestinalnematodes,includingHaemonchuscontortus.Thismodifiedmethoduses onlyliquidaxenicmedium,insteadofagarplatesinoculatedwithEscherichiacoli,andtwo selectivesievestoobtainadultnematodes.Duringscreening,theuseofeitherbalancedsalt solution(M-9)ordistilledwaterresultedinaveragesof99.7(±0.73)%and96.36(±2.37)% motileadults,respectively.AdultwormstoleratedDMSO,ethanol,methanol,andTween 80at1%and2%,whileLabrasol®_{(abioenhancerwithlowtoxicitytomammals)andTween}

20weretoxictoC.elegansat1%andwereavoidedassolvents.Thehighavailability,easeof culture,andrapidproliferationofC.elegansmakeitausefulscreeningsystemtotestplant extractsandotherphytochemicalcompoundstoinvestigatetheirpotentialanthelmintic activityagainstparasiticnematodes.

Published by Elsevier B.V.

1. Introduction

Caenorhabditis elegansis a free-livingnematode nat-urallyfoundintemperateclimatesoils.Experimentation withthisnematodebeganin1960whenresearcherswere lookingforamulticellularorganism,withafewcells,easy

∗ Correspondingauthor.Tel.:+13042562827;fax:+13042562921.

E-mailaddress:Jorge.Ferreira@ars.usda.gov(J.F.S.Ferreira).

toraiseandreproduceforembryonicdevelopmental stud-ies.Since then, C. eleganshasbecome one of the most studiednematodesinmanyareasofbiology.

TheOrderRhabditida,towhichC.elegansbelongs,is closelyassociatedwiththeOrderStrongylida,which con-tainstheimportanttrichostrongyleparasitesofruminants, includingHaemonchuscontortusandTrichostrongylusspp. Therhabditidandstrongylidnematodeshavebeenplaced inCladeVbasedongeneticanalysis.Othercommon nema-todes of domestic animalsand humansare less closely

0304-4017/$–seefrontmatter.Published by Elsevier B.V.

relatedandhavebeenplacedinotherclades.Forexample, ascaridandfilarialwormsareinCladeIII,andTrichinella andTrichurisinCladeI(GearyandThompson,2001).

SimpkinandColes(1981)examinedtheeffectof com-mercialanthelminticsusingC.elegansasanexperimental modelandconcludedthatthis nematodesatisfiesmany of the criteria needed for an invitro test because it is cheap,readilyavailable,andeasytoworkwith.Sincethen, otherparasitologistshavealsousedthismodeltoscreen anthelminticdrugs(McGawetal.,2007).Besidesthe nema-tocidaleffect,themodeofactionofanthelminticdrugscan beevaluatedinvitrothroughnematodebehavior, locomo-tion,andreproduction.IftesteddrugsareeffectiveinC. elegansculturesatlowconcentrations,itisreasonableto assumethattheymayhaveanthelminticactivityagainst relatednematodes,includingH.contortus(Thompsonetal., 1996).

Gastrointestinal parasitism is a serious problem in small ruminant production due to high morbidity and highmortalitycausedbyH.contortusandrelated nema-todes.Thisproblemhasbeenaggravatedbythegrowing reportsofmulti-drugresistantgastrointestinalparasites worldwide (Jackson and Coop, 2000; Zajacand Gipson, 2000;Kaplan,2004).Thebesttesttodetermineifa com-poundhasanthelminticactivityforveterinaryusewould betouseinfectionsinthenatural ruminanthost. How-ever,thisrequires livestockfacilitiesandlargeamounts ofplantmaterial,makingextensivescreeningnotfeasible. Tofacilitateinitialscreeningofproducts,severalinvitro assays have been used extensively to assess potential anthelminticactivityofnaturalcompounds,includingthe egghatchassay,thelarvalmotilityassay,andothers(Hoste etal.,2006;Egualeetal.,2007;Brunetetal.,2008). How-ever,theseassaysdonotinvolvetestingproductsagainst adultstages,which are themostharmfultothe defini-tivehost.TestswithadultH.contortusinvolvethekilling anddissectingof ananimalhosttoprovidesubjectsfor theadultwormmotility(AWM)assay(Marie-Magdeleine etal.,2009).UsingaC.elegansmodelforscreeningprovides theadvantages ofa lowcostinvitrolaboratorymethod combined withthe ability toexamine activityof com-poundsagainstadultparasiticstagesinrelatednematode species.Theobjectivesofthisworkweretotestamodel systemusingliquid,axenicculturesofC.elegansto(1) prop-agatetheorganism,(2)select(withsieves)adultwormsfor testing,and(3)evaluatedifferentsolventsfortheir toler-abilitytoC.elegans.Thisimprovedmethodisdesignedto screenplantextractsandcompoundsfortheiranthelmintic activity.

2. Materialsandmethods

2.1. Solvents

Dimethyl sulfoxide (DMSO), ethanol, methanol, ace-tone were all reagent grade (Fisher Scientific, www. Fishersci.com). Labrasol®_{, a bioenhancer composed of} caprylcaproylpolyoxyl-8glyceridesofbothvegetableand petrochemicaloriginwasdonatedbyGatefossé(Paramus, NewJersey,USA),andbothTween20andTween80were providedbySigma–Aldrich(www.Sigma-Aldrich.com).

2.2. MaintenanceofC.elegans

StrainN2(wildtype)acquiredfromtheUSDA Nema-tologyLaboratory,Beltsville,MD,wasraisedinanaxenic culture medium (Chitwoodand Feldlaufer, 1990), com-posedof90mldistilledwater,3.0gyeastextract(catalog No.Y1625,Sigma–AldrichCorp.,St.Louis,MO),3.0gsoy peptone (Sigma P-0521), 1.0g dextrose,0.25ml choles-terol(Sigmacat.No.C8667)solution(5mgcholesterolper 1.0ml95%ethanol).Themediumwasautoclavedandthen supplementedwith10mlofahemoglobinstocksolution containing 0.5%hemoglobin (Sigmacat. No.H-2500) in 100ml 0.001MKOH filter-sterilized through a 0.45␮m

sterile filter, then through a 0.22␮m sterile filter, and

frozenuntilneeded.Wormsweresub-culturedeachweek bytransferringtwodropsfromaone-week-oldculturetoa sterilescintillationvialcontaining1.0mloffreshmedium, andincubatedat24◦_C.

2.3. Isolationofadultnematodes

Nematodeswereidentifiedasadults,youngadults,L4 orjuveniles(L1–L3)bysizeandthepresenceandextent of vulvar development(Wood, 1988). The L1–L3 stages wereidentifiedbytheirsmallersize.Thedevelopmentof thevulvainyoungadultsandadultswasobservedwith aninvertedmicroscope.TheL4lackedthevulvaandonly presenteda clearareawithasemi-circularshapeinthe genitalareamidwayalongthelengthofthenematode. Dur-ingthechangefromL4toadultstage,thevulvabecomes prominent(youngadult)andtheclearareadisappears(Bull etal.,2007).Thetestswereperformedwithyoungadults andadultswithintactcuticle.Youngadultsare morpho-logically similartoadults, butsmaller, and do not bear eggs.

Inordertoselectonlyyoungadultsandadults,fourvials containing1.0mlofC.elegansaxenicculture5–7-dayold weretransferredtoa50-mltubeandthevolumewas com-pletedwithsteriledistilledwater.Allstagesofthelifecycle werepresentandthetubewasplacedinarackfor3minto allowsettlingofthelargestnematodes.Thesurfacewater containingsmallerfloatingnematodeswasthenremoved toreducethevolumetoapproximately0.5ml.Nematodes wereseparatedaccordingtotheirsizeusingtwosieves. Sieveswerecreatedbyinsertingnylonfilterclothbetween 2piecesofPVCpipeof2.1and1.9cmdiameter.First,we usedasterile38␮msievetoretainthelargestnematodes

andallowsmallernematodestomigratethroughthesieve. This procedureretained youngadults, adults,and large deadnematodes,whichwerenon-motileandstraightened (Fig.1).Approximately10mlofdistilledwaterwasusedto facilitatethemigration.

Nematodesretained in the38-␮msievewere

trans-ferredwith50mlsterilewaterontoalargermesh53-␮m

sieve.Inthisprocedureallactivenematodes(adultsand youngadults)passedthroughthemeshand alldeador inactive nematodes were retained by the 53-␮m sieve

(Fig.2).

Fig.1.Adults,youngadultsanddead(straightened)nematodesretainedbythe38-␮mmeshsieve.Smallnematodes(L4andsmaller)werenotretained bythissieve.ApproximatelengthsofyoungadultsandlateadultsraisedonagarplatesseededwithE.coliwere1.0mmand1.4mm,respectively(Knight etal.,2002).Ournematodes,raisedentirelyinliquidcultures,wereapproximately40%longerthanagarplate-raisedones.Basedonthisinformation,the approximatemagnification=28×.

Thefinal solutionhada concentrationofapproximately 50nematodes/20␮l.

2.4. Assaypreparation

Tests wereperformed usinga balanced salt solution (M-9) as the diluent for solvents and M-9 was also used asthemedium topreparea nematodestockwith 50nematodes/20␮l.M-9solutionwascomposedof1.5g

KH2PO4,3gNa2HPO4,2.5gNaCl,0.5ml1MMgSO4,and sterile distilled water to bring the volume to 500ml (Brenner,1974).Testswereperformedin24-wellplates containingatotalvolumeof250␮l/well,with6replicates

pertreatment.The24-wellplateswerecoveredwith trans-parentplastic, andincubated at24◦_{C for 24h. TheM-9} medium produced betternematode survival than using

distilledwater,perhapsbecausethemediumbetter pre-servedthenematode’sosmoticbalance.

2.5. Assayevaluation

After incubation at 24◦_{C for 24h, plates were read} usinganinvertedmicroscopeandallnematodescounted anddeterminedasmotileornon-motile.Theywere con-sideredmotilewhentheyexhibitedanymovement,and as non-motile when there were no tail, head, or pha-ryngeal movements during 5s of observation (Skantar et al., 2005). It is important to differentiate motilityin adultnematodesfrommovementcausedbylarvaehatched fromeggsinside thebodyof deadC.elegans.The nega-tivecontrol groupconsistently showed95–100%motile nematodesandthepositivecontrol(levamisole)0%motile nematodes24hafterincubation.Tofacilitatecountingof

Table1

Effectofsolvents(DMSO,ethanol,methanol,acetone,Labrasol®_{,Tween20andTween80)intheadultC.elegansmotilitytest.Resultsaretheaverages} (%)oftwotests,withsixreplicateseach,ofmotilewormsobservedateachsolventconcentration.Theaverage%motileadultlarvaefortheM-9control mediumwas99.70±0.73%24hafterincubation.Standarderrorsarepresentedinbetweenparenthesis.

Solvent Concentration

1% 2% 4% 6%

DMSO 91.89(2.07) 82.86(3.88) 45.74(5.23) 33.77(2.76) Ethanol 95.85(5.15) 87.45(5.34) 82.32(3.17) 44.05(6.27) Methanol 90.76(3.28) 81.11(3.1) 83.98(2.72) 54.08(2.15) Acetone 90.71(4.67) 82.52(3.19) 73.58(2.2) 56.25(3.89)

Labrasol 0 0 0 0

Tween20 29.55(4.73) 9.23(1.47) 9.28(3.81) 9.53(1.55) Tween80 94.28(3.03) 94.10(5.33) 70.30(2.45) 44.14(2.54)

50nematodes/well,horizontallinesweredrawnatthe bot-tomoftheplatesat0.1mmdistanceintervals.

3. Resultsanddiscussion

OurworkindicatesthatthemethodusingliquidC. ele-gansculturesisafastandreliablewaytopropagateand synchronizeC.elegans.Thismethodcanbeusedtoscreen crude plantextracts and compoundsfor their potential anthelminticactivity.Themethoddiffersfrompreviously describedscreeningtests(Stiernagle,2006)inthatituses: (1)anaxenic(noE.coli)liquidmediumthroughout,instead ofagarplatesinoculatedwithE.colitomaintainand propa-gateC.elegans,(2)nylonsievesof38and53␮mporesizeto

selectadultnematodesfortesting,insteadoflifecycle syn-chronization,and(3)onlyearlyadultandadultnematodes, ratherthanmultiplestages,tocarryoutthetests(Fig.2). ThemainadvantagesofavoidingagarplateswithE.coli lawnsare:(1)timeandeffortsavedonplatepreparation andinoculationwithE.coliasafoodsourceforC.elegans,(2) reductionincontaminationandeliminationofantibiotics duringthescreeningtests,and(3)preventionofbacterial metabolismofphytochemicalsfromtheextracts(Simpkin andColes,1981).Additionaltimewasgainedfromselecting wormsthroughsievingandavoidingthesynchronization process.

Duringthedevelopmentofthismethod,wenoticedthat wormscouldtolerateupto2%DMSO,ethanol,methanol, acetoneorTween80withoutlossofmotility,but2%Tween 20and1%Labrasol(Gatefoseé,France)wereverytoxicto C.elegans(Table 1).However,even2%DMSO orTween 80couldnotdissolvelyophilizedextractsmadewithlow polaritysolvents(e.g.,dichloromethane,chloroform,pure ethanol) or non-polar solvents (e.g., hexane, petroleum ether).Thus,thesesolventsshouldbeavoidedforextract preparation.ThebioenhancerLabrasol®_{wasveryeffective} indissolving lyophilizedplant extractsfor anthelmintic testingwithouttoxicity ingerbilsat25%(Squiresetal., 2011),andinmiceat66%(Ribnickyetal.,2009). Unfortuan-tely,LabrasolkilledallnematodesintheC.elegansinvitro systemevenat1%(Table1)andcouldnotbeusedforplant extractdissolution.

ThehighsurvivalratesofC.elegansin1%DMSO(91.89%), ethanol(95.85%),methanol(90.76%),acetone(90.71%),and Tween80(94.28%),indicatethatanyofthesesolventsused ataconcentrationof1%or2%(Table1),canbeusedto

helpdissolvecrudeplantextractswithlowtoxicitytoC. elegans.However,oneshouldconsidersurvivalratesof sol-ventcontrolswhencalculatingsurvivalratesinresponseto testedplantextracts.Thiswillpreventanover-estimation of anthelminticactivity of the plant extracts. Although datarelatedtotestswithplantextractsindifferentmedia are not presentedhere,it is important tomentionthat theaxenic(basal)mediumcontaininghemoglobinandsoy peptoneisagoodmaintenancemedium,buttheseproteins reactwithtanninsmakingitunsuitableforscreening.Also, whilebothM-9anddistilledwaterbothresultedinaverage motileadultsof99.7(±0.73)%and96.36(±2.37)%, respec-tively,nematodesseemedmoreadjusted totheisotonic M-9mediumthanondistilledwater,withtheformerbeing bettersuitedforplantextractscreening.

4. Conclusion

C. elegans cultured in medium containing heme (hemoglobin),replacestheuseofagarplateswithanE.coli lawn.Whilethisaxenicculturemethodisasimplerand fastermethodforprovidinglargenumbersofclean,active adultnematodes,theisotonic M-9mediumhasno pro-teinandwasmoresuitabletokeepC.elegansforscreening purposes.Byusingtheselectivesievesdescribedhere,itis possibletocollectmanyadultswithinoneweekinasmall volumeofmedium.Freezingtheculturemediuminsmall volumesmakesitrapidlyavailablewhenneeded,inany quantity,comparedtopreparationofE.coliinoculatedagar plates.Asaresult,onepersoncouldscreenatleastsixtests withresultsavailablein48hinsteadof72–96husingagar plateswithE.coli.

Roleoffundingsources

The funding sources had no influence in the study design, collection,analysis,interpretationofdata,inthe writingofthemanuscript,orinthedecisiontosubmitthe manuscriptforpublication.

Conflictofintereststatement

oversimilarproducts.TheUSDAisanequalopportunity providerandemployer.

Acknowledgements

Theauthorsaregratefulforthefinancialsupportfrom CAPES-Brazil and for thepartial supportprovided bya specificcooperativeagreementbetweentheAppalachian FarmingSystemsResearchCenter(USDA-ARS)andthe Vir-giniaPolytechnic InstituteandStateUniversity(Virginia Tech).WegreatlyappreciatethesupportofDr.David Chit-wood(USDA-ARSNematologyLab)andhistechnicaland scientificstaffattheonsetofthiswork.Wearealso grate-fulfortheencouragementfromDr.DavidBeleskyandthe effortsofMr.MarcPeele(AFSRC,USDA-ARS)withC.elegans cultures.

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