TurkJZool 26(2002)111-119 ©TÜB‹TAK ResearchArticle EffectsofAntimicrobialAgentsontheSurvivalandDevelopmentof LarvaeofPimplaturionellae L.(Hymenoptera:Ichneumonidae) RearedonanArtificialDiet KemalBÜYÜKGÜZEL DepartmentofBiology,FacultyofScience-Art,KaraelmasUniversity,67100,‹ncivez,Zonguldak-TURKEY fievkiYAZGAN DepartmentofBiology,FacultyofSciences,AnkaraUniversity,06100,Beflevler,Ankara-TURKEY Received:28.06.2000 Abstract: Theeffectsof13antimicrobialagentsthathavedifferentstructuresandmodesofaction,onthesurvivalanddevelop- mentofthehymenopterousendoparasitoid, Pimplaturionellae L.,wereinvestigatedbyrearingthelarvaeasepticallyonchemically definedsyntheticdiets.Theseeffectsvariedaccordingtotheirkindanddietarylevels.Theagentstestedexertedtheireffec tsgen- erallyduringthepost-larvaldevelopmentoftheinsect.Thepost-larvalsurvivalanddevelopmentwerenegativelyaffectedbym ost oftheagentstestedatcertainlevels.Theantimicrobialagentstestedalsoaffectedthelarvaldevelopmentbuthadnosignifi cant effectsonthelarvalsurvivaloftheinsect.Thelarvaeshowedawidetoleranceagainstboththekindanddietarylevelsoftheagents. Noneoftheagentstestedweretoxictothelarvae.Sodiumbenzoateandnystatinatlowlevelshadpositiveeffectsonthefoodcon- sumptionofthelarvae. KeyWords: Pimplaturionellae,Antimicrobialagents,Insect,Endoparasitoid Baz›AntimikrobiyalAjanlar›nSentetikBesindeYetifltirilen Pimplaturionellae L. (Hymenoptera:Ichneumonidae)Larvalar›n›nYaflamaveGeliflmesineEtkileri Özet: Endoparazitoidzarkanatl›türü, Pimplaturionellae L.,kimyasalyap›s›bilinensentetikbesinortamlar›ndaaseptikflartlarda beslenerek,farkl›yap›veetkiyesahiponüçantimikrobiyalajan›nböce¤inyaflamavegeliflmesineetkileriincelendi.Buetkiler dene- nenantimikrobiyalajan›nçeflidinevemiktar›nagörede¤iflmektedir.Denenenajanlar›netkileriço¤unluklalarvasonras›evreler de ortayaç›km›flolupbuevrelerdekiyaflamavegeliflmeolumsuzyöndeetkilenmifltir.Antimikrobiyalajanlarayn›zamandalarvalevr e- dekigeliflmeyideetkilemiflancakbuevredekiyaflamaüzerindeönemlibiretkiyapmam›flt›r.Larvalarantimikrobiyalajanlar›nhe m çeflidinehemdebesinselmiktar›nakarfl›geniflbirtoleransgöstermifltir.Denenenajanlar›nhiçbirilarvalarüzerindetoksiket kiyap- mam›flt›r.Sodyumbenzoatvenistatinindüflükmiktarlar›larvalar›nbesintüketimineolumluetkidebulunmufltur. AnahtarSözcükler: Pimplaturionellae,Antimikrobiyalajanlar,Böcek,Endoparazitoid Introduction eggtoadultinstarinlaboratoryconditions(1-9). Becauseoftheirpredatorynature,parasitic Chemicallydefineddietsthathavebeendevelopedtorear hymenopterousspeciescanbeusedaspotentialtoolsin parasiticinsectsarehighlysuitableforthedevelopment biologicalcontrolprograms.Manyattemptshavebeen ofmicroorganisms.Variousantimicrobialagentswere madetousetheseinsectsintheseprograms.Oneofthese usedininsectdietstopreventmicrobialcontamination wastocultivatetheseinsectspermanentlyinalaboratory. andpropagation(2,10-16),butaverylimitedamountof Todoso,somechemicallydefinedsyntheticdietsand workhasbeendoneontheeffectsoftheseagentson rearingtechniquesweredevelopedforsomespecies.By parasitichymenopterousspecies(17-20).Some usingthesedietsandtechniques,someparasitic hymenopterousinsects,especiallysocialones,produce hymenopterousspeciesweresuccessfullyrearedfrom somenaturalsubstanceswhichfunctionasantimicrobial 111 EffectsofAntimicrobialAgentsontheSurvivalandDevelopmentofLarvaeof Pimplaturionellae L.(Hymenoptera:Ichneumonidae) RearedonanArtificialDiet agents(21-25).Forexample,hymenopterousparasitoid ofanaminoacidmixture,lipidmixture,aninorganicsalt P.turionellae larvaedischargesuchasubstanceintotheir mixture,glucose,RNAandamixtureofwater-soluble host.Thisactsasanantimicrobialagentduringthelarval vitamins.Themethodsusedinthepreparationofstock developmentoftheinsect(26).Someantimicrobial solutionsofdietaryingredients,thedietfromthese agentscanpermanentlyortemporarilychangethemode stocks,thesterilizationtechniques,theaddingof ofreproductionofthethelytokyformofsomeparasitic sterilizedvitamin-glucoseandantimicrobialagents hymenopterousinsectstothearrhenotoky,whichisa solutiontothediet,thedispensingofthedietsintotest- normalsystemofreproductioninthehymenoptera(27- tubes,theobtainingofthesterilizedlarvaeandtheir 32).Theseindicatetheimportanceofantimicrobial inoculationontodiets,andthepreparationofgelatine agentsinthedevelopmentofhymenopterousinsects. capsulesforpupationweremostlythesameasthose Thisworkisimportantinunderstandingthenutritional describedelsewhere(20). suitabilityoftheantimicrobialagentsasadietaryadditive Someoftheantimicrobialagentsusedinthiswork substanceintheartificialrearingoftheseinsects.Such suchastetracyclinehydrochloride(968µg/mg), workmightalsobeofgreatpracticalimportanceinthe chloramphenicol(980µg/mg),erythromycin(Freebase, controlofbacterialandfungalcontaminations,whichare averyseriousprobleminartificialdietsforthemass- 820µg/mg),trimethoprim(99%),cycloheximide(98%) rearingof P.turionellae andotherpupalendoparasitoid andsodiumbenzoate(99%)wereobtainedfromSigma hymenopterousinsects.Contaminationisoftenamajor ChemicalCo.Theotherswereobtainedfromdifferent factormakingtheirrearingunsatisfactoryandsometimes firmssuchaspenicillinG(potassiumsalt,1600units)and impossible.Thisproblemintheartificialrearingsystem streptomycinsulphatefromClaus&HuthLtd.,methylp- ofourinsectmostlycausestimeconsumptionand hydroxybenzoate(pure,crystalline)fromKoch-Light extravagancyofnutrientchemicals.However, Pimpla Lab.,rifampicin(-SV,95%)fromKoçakMedicineCo., larvaeovercometheproblembydischargingananal lyncomycinhydrochloride(877µg/mg)fromEczac›bafl› secretionwhichhasantibioticfunctionduringtheir MedicineCo.,andcephradinemonohydrate(942µg/mg) developmentintheirhoststoprotectthemselves(26).To andnystatin(5300units/mg)fromBristol-MyersSquibb rearhealthyadultsforbiologicalcontrol,itisnecessaryto Inc.Mostoftheantimicrobialagentstestedinthiswork controlmicrobialcontaminations.Someworkhasbeen wereselectedfromcommonlyusedonesinthelarval doneonantifungalagentsinartificialdietsbutverylittle dietsforartificialrearingofsomeentomophagousinsects dealswithantibioticsorevenwithpupalendoparasitoids (12,17-20,33,34).Inadditiontotheseagents, Theworkwhichhasbeendonedealswiththe trimethoprim,lincomycinandcephradine,whichhavenot individualeffectsofsomeantifungalagentsand beenpreviouslyusedinanyartificialrearingdiet,were antibioticsonthesurvivalanddevelopmentofthelarvae alsotested.Thus,itwasplannedtotestoneortwo ofapupalendoparasitoidhymenopterousinsect, Pimpla agentsfromeachgroupofantimicrobialagentswith turionellae,whichisapotentialbiologicalcontrolagentof differentstructuresandmodesofaction.Ourprevious manylepidopterouspestinsects.Therefore,wetriedto workshowedthatsomeantimicrobialagentstestedsuch determinethelevelsoftheseantimicrobialagentswhich aspenicillinandstreptomycinexertedtheirmaineffects haveminimalornodetrimentaleffectsontheinsect. atalevelof50mgof100mlofdiet,whereasrifampicin atthislevelwashighlyeffectiveonthepost-larvalsurvival anddevelopmentofP.turionellae (20).Therefore,inthe MaterialsandMethods presentwork,thetestedlevelsoftheantimicrobial P.turionellae adultsusedintheexperimentswere agentswereconsideredtocenterbeloworaroundalevel rearedinthelaboratoryonthepupaeofthegreaterwax of50mgof100mlofdiet.Somepreliminary moth,Galleriamellonella L.,at23±1ºC,75±5%r.h.and experimentswereconductedtodeterminetherangeof witha16hilluminationperiod.Adultswerefeddaily the13agents.Basedontheresultsoftheseexperiments, with50%honeysolutionand G.mellonellae pupae penicillinandstreptomycinatlevelsof10,20,30and40 hemolymph. mg;rifampicin,methylp-hydroxybenzoateandsodium ThesyntheticdietdescribedbyYazgan(3)wasused benzoateatlevelsof2.5,5.0,7.5,10.0mg; forrearingthelarvaeofP.turionellae.Thedietconsisted cycloheximideatlevelsof0.002,0.004,0.006,0.008 112 K.BÜYÜKGÜZEL,fi.YAZGAN mg;andtherestoftheantimicrobialagentsatlevelsof reachfifth,pupalandadultinstars)andsurvival(number 15,30,45,60mg/100mlofdietweretested.Thus, ofsurvivorsintheseinstars)ofthelarvae.Dataonthe fourlevelsofeachagentweretestedandcomparedwith rateofdevelopmentwereevaluatedbyanalysisofthe acontrol(withoutantimicrobialagents).Onlybidistilled variance(35).Todeterminesignificantdifferences waterwasusedasthediluentforalltheantimicrobial betweenmeans,Duncan’s(36)multiplerangetestwas agents.Weobtainedaquitestablesuspensionby used.DataonsurvivalwerecomparedbyX 2 test(35). vigorouslyshakingthemixtureinthepreparationof WhenFandX2 exceededtheir0.01value,thedifferences solutionsoflowsolubleagentsinwatersuchasnystatin, wereconsideredsignificant. rifampicinandmethyl p-hydroxybenzoate.Nospecial solventsorprotectivesubstanceswereusedfordilution ofsuchagentstoavoidpossiblechangesinthechemical Results andphysicalcompositionofthedietsandthustoprevent Theeffectsofthemosteffectiveantimicrobialagents theirpossiblecontributiontotheeffectsofantimicrobial onthesurvivalanddevelopmentof P.turionellae larvae agentsontheinsect. areshownintheTable. Theagentstestedwereexpressedasmg/100mlof Thelowestlevelofrifampicinhadnosignificanteffect diet.Theirdesiredamountsweredissolvedinbidistilled oneithersurvivalordevelopment,but5mgand10mg water,thensterilizedthrough0.22µmpore-sized ofitsignificantlydecreasedthepostlarvalsurvival.The membranefilterandaddedtothedietbeforegel adultdevelopmentoftheinsectwasalsosignificantly formation.Thevolumeofthedietwasestablishedby retardedbythedietwhichcontainedonly5mgof adjustingtheamountofwaterinthediet.Onenewly rifampicin.Thehighestlevelofthisantibioticalso hatchedlarvawasinoculatedintoeachtest-tube retardedthelarvaldevelopmentbyapproximatelyfour containingabout0.5mlofdietwithincreasinglevelsof days.Chloramphenicol,atalltestedlevels,didnotaffect