Lethal and sublethal effects of insecticides on mortality, migration and host searching behaviour of tersilochine parasitoids on winter oilseed rape

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1 Lethl nd sublethl effects of insecticides on mortlity, migrtion nd host serching behviour of tersilochine prsitoids on winter oilseed rpe Disserttion zur Erlngung des Doktorgrdes der Fkultät für Agrrwissenschften der Georg-August-Universität Göttingen vorgelegt von Ndine Neumnn geboren in Freckenhorst Göttingen, Jnur 2010

2 D 7 1. Referent: Prof. Dr. Stefn Vidl 2. Korreferent: Prof. Dr. Stefn Schütz Tg der mündlichen Prüfung:

3 Contents Tble of contents pge Chpter I Generl introduction 1 Studied insect pests nd their prsitoids on oilseed rpe 2 Host loction by prsitoids 3 Possible effects of insecticides on prsitoids 6 Strtegies for conservtion of prsitoid popultions in integrted pest mngement systems 7 References 8 Chpter II Role of voltiles emitted from the host-plnt complex in host loction by the cbbge stem borer prsitoid Tersilochus obscurtor on winter oilseed rpe 15 Abstrct 15 Introduction 15 Mteril nd Methods 16 Results 22 Discussion 32 Acknowledgement 37 References 37 Chpter III Effect of insecticide residuls on host finding cues of Tersilochus obscurtor, the key prsitoid of cbbge stem weevil 42 Abstrct 42 Introduction 42 Mteril nd Methods 44 Results 47 Discussion 57 Acknowledgement 61 References 61 Chpter IV Effect of insecticides on prsitism of stem mining pests nd migrtion of tersilochine prsitoids on winter oilseed rpe (Brssic npus L.) 65 Abstrct 65 Introduction 66 Mteril nd Methods 67 Results 69 Discussion 77 References 80 Chpter V Impct of insecticide tretments on mortlity, prsitism nd host loction of prsitoids ttcking pollen beetle lrve 85 Abstrct 85 Introduction 86 Mteril nd Methods 88

4 Contents Results 90 Discussion 101 References 105 Chpter VI Generl discussion 109 Host serching 109 Lethl nd sublethl effects of insecticides on prsitoids 112 Impct of insecticides on prsitism on oilseed rpe fields 114 Strtegies for conservtion of prsitoids 116 References 118 Summry 124 Acknowledgement 127 Curriculum vite 128

5 Chpter I Generl introduction Oilseed rpe (Brssic npus L. vr. oleifer Metzg.) (Brssiccee) is the third most commonly grown crop in Europe (FAO, 2007). In Germny, oilseed rpe is cultivted on 1.46 Mill h in 2009/2010 (UFOP, 2009). It is grown for oil, for humn consumption, s well s for renewble energy resources nd niml food. With the extention of the plnted re in the lst twenty yers, dmge by pests nd diseses of oilseed rpe becme more severe (Alford et l., 2003; Lmb, 1989). One of the most importnt limiting fctors is the lrge vriety of pest insects ttcking the oilseed rpe crop (Alford et l., 2003). As result brod-spectrum, non-selective insecticide compounds re pplied in utumn, spring nd summer for control these economiclly-importnt pests (Menzler-Hokknen et l., 2006). They re often pplied prophylcticlly without regrd to pest control thresholds. Frequent pplictions of insecticides hve cused widespred resistnce of pollen beetle popultions ginst pyrethroid compounts in mny Europen countries, including Germny (Heimbch et l., 2006), Polnd (Wegorek & Zmojsk, 2006) nd Denmrk (Hnsen, 2003). Therefore, the integrtion of nturl regultory mechnisms on pest popultions, such s nturl enemies, becomes more importnt. Minimizing the hrmful side-effects of insecticide ppliction on nturl enemies is one of the min objectives in IPM sttegies. Therefore, optimiztion of insecticide use, e.g. product selection nd timing of ppliction, while minimizing negtive effects on beneficil rthropods (Flint & Bosch, 1981). There is little informtion on side-effects of chemicl insecticides on prsitoids of pests insects on oilseed rpe. The min objectives of this study re: 1. to investigte the role of voltile production by uninfested oilseed rpe plnts nd by plnts infested by lrve of C. pllidctylus in behviourl nd electrophysiologicl biossys nd the effect of insecticide tretments 2. to study the impct of insecticide tretments on prsitism nd migrtion of prsitoids ttcking stem weevil nd pollen beetle lrve in field experiments 3. to nlyse lethl nd sublethl effects of insecticides on prsitoids in lbortory experiments 4. to determine whether the side-effects of insecticides on hymenoptern prsitoids on winter oilseed rpe cn be minimized by optimiztion of insecticide uses, such s product selection, timing of ppliction nd reduction of field dose rtes 1

6 Chpter I Studied insects pests nd their prsitoids on oilseed rpe Stem-boring weevils Two stem-mining pests of oilseed rpe were investigted in this study, the cbbge stem weevil Ceutorhynchus pllidctylus (Mrshm) nd the rpe stem weevil Ceutorhynchus npi Gyllenhl (Coleopter: Curculionide). Both species re univoltine. Adults of rpe stem weevil migrte to crops of oilseed rpe in erly spring (Februry/Mrch). Femles ly their eggs singly into the stems nd the lrve htch fter one or two weeks. The lrve mine within the stem for 3-5 weeks before they leve the plnt to pupte in the soil. Adults overwinter in n erthen chmber in the soil nd emerge the following spring (Schmutterer, 1956; Debouzie & Bllnger, 1993; Pul, 2003). Cbbge stem weevils migrte from their overwintering hbitts (in lef litter t the edges of wood or shrubs) to oilseed rpe in spring (Mrch/April) (Alford et l., 2003). Femles ly their eggs in btches of 4-6 eggs (Winfield, 1992) into lef petioles (Brri et l., 2005; Ferguson et l., 2003). First nd second instr lrve feed within the pith of petioles, third instr lrve migrte to the min stems (Brri et l., 2005). Mture lrve drop to the soil for puption (Broschewitz & Debler, 1987). Depending on wether conditions, puption tkes bout dys (Broschewitz, 1985). New-genertion dults emerge from soil t the end of June or beginning of July. After feeding on vrious species of Brssiccee they migrte to their overwintering sites (Broschewitz & Debler, 1987). The key lrvl endoprsitoids of rpe stem weevil nd cbbge stem weevil re Tersilochus fulvipes (Grvenhorst) nd Tersilochus obscurtor Aubert (Hymenopter: Ichneumonide), respectivelly (Ulber, 2003). They re univoltine nd koinobiont. Adult prsitoids migrte to oilseed rpe crops in spring (Mrch/My) from fields where oilseed rpe crop hs been grown the previous yer. Femles ly their eggs singly into the host lrv. The prsitoid lrv remins in its first instr until the host lrv is mture nd hs left the plnt to pupte in the soil; then it develops rpidly nd kills the host prepup. The dult prsitoid dipuses in its pupl cocoon in soil nd emerges the following spring (Lehmnn, 1969; Nissen, 1997; Ulber, 2003). Pollen beetle The pollen beetle, Meligethes eneus (Fbricius) (Coleopter: Nitidulide), is n univoltine pest on oilseed rpe. The over-wintered dult beetles migrte into oilseed rpe fields in spring nd oviposit into buds of 2-3 mm size. The dults nd the two lrvl instrs feed on buds nd flowers. The development from the egg to the dult stge tkes bout dys (Nilsson, 2

7 Chpter I 1988). Mture lrve drop to the ground nd pupte in soil. New-genertion beetles emerge in summer nd feed on flowers before hiberntion strts in August (Hokknen, 1993). Adults overwinter in field mrgins, woodlnd nd hedgerows. The oilseed rpe crop is most susceptible to pollen beetle dmge t the erly bud stge nd becomes less sensitive s the crop develops (Alford et l., 2003). The key prsitoids of pollen beetle in Centrl Europe re Phrdis interstitilis (Thomson), Phrdis morionellus (Holmgren), nd Tersilochus heterocerus Thomson (Hymenopter: Ichneumonide) (Nilsson, 2003). These univoltine, koinobiont endoprsitoids re specilised on Meligethes spp.. The prsitised host lrv continues its development until mturtion. Lrvl development nd puption of the prsitoid tkes plce inside the host prepup. Adults overwinter in cocoons in soil nd emerge during the following spring (Osborn, 1960). There is niche segregtion between the prsitoid species: P. interstitilis oviposits primrily into beetle eggs within green buds, P. morionellus ly their eggs into lrve within buds nd open flowers nd T. heterocerus oviposits into second instr lrve within flowers (Osborn, 1960; Winfield, 1963; Nilsson, 2003). Reports in the literture provide evidence tht mortlity due to prsitism my be sufficient fctor to void grdtion of pest popultion on oilseed rpe (Büchi & Roos-Humbel, 1991; Sedivy, 1983; Alford et l., 2003). Prsitiztion rtes of rpe stem weevil lrve by T. fulvipes between 1% nd 21 % were evluted in Germny (Ulber, 2003), while higher prsitiztion rtes were reported in studies from Frnce nd Czechoslovki (Jourdheuil, 1960; Sedivy, 1983). Levels of prsitism of C. pllidctylus by T. obscurtor between 19.6% nd 52.5% were found t Goettingen (Klingenberg, 1991; Nitzsche, 1998; Ulber, 2003) nd in Frnce (Jourdheuil, 1960). Prsitiztion rtes of M. eneus rnged from 16% to 83% in Sweden (Nilsson & Andresson, 1987), Germny (Nissen, 1999) nd Switzerlnd (Büchi & Roos-Humbel, 1991). Prsitiztion rtes of up to 50% re likely to hve n influence on pest bundnce in the long run (Klingenberg & Ulber, 1994). Hokknenen et l. (1988) reported tht in some res of Finlnd where the prsitiztion rtes of pollen beetle were s high s 60% to 80%, the need for chemicl control ws by fr less importnt thn in res with low prsitiztion rtes. Host loction by prsitoids Plnts defend themselves ginst herbivores by using chemicl nd physicl trits tht directly ffect herbivore performence or indirectly through trits tht ttrct nturl enemies 3

8 Chpter I (Tkbyshi & Dicke, 1996; Pré & Tumlinson, 1999). Voltile orgnic compounds (VOCs), specificlly those relesed following herbivory, re known to ttrct nturl enemies. Key hymenoptern prsitoids of mjor pests on oilseed rpe re highly specific. Their serch for hosts is described s, (i) loction of the hbitt of the host (the crop) (ii) loction of the host within the hbitt nd (iii) cceptnce of the host (Vinson, 1998). Prsitoids of oilseed rpe use upwind nemotxis to locte the oilseed rpe crop with their hosts (Willims et l., 2007). Generlly, prsitoid species re lso ttrcted to odours of undmged plnts (Kiser & Crdé, 1992) nd/or rtificilly dmged plnts (Mtticci et l, 1994; Potting et l., 1999), but these cues re reltively unrelible in indicting host presence. Vrious nturl enemies re known to discriminte between voltiles emitted by uninfested nd herbivore-infested plnts. When plnts re ttcked by herbivores, they my emit specific compounds tht re not produced fter rtificil dmge (Turlings et l., 1990; Dicke et l., 1990b; Pré & Tumlinson, 1997). Chemicl stimuli ementing from the host-plnt complex cn originte from the herbivore, the plnt, or from combintion between herbivore nd the plnt. Although odours emitted from hosts or host products re very relible indictors for host presence their long-rnge detectbility is very low (Turlings et l., 1991; Vet et l., 1991). Plnt voltiles produced in response to herbivore dmge my contin informtion on the identity of the host. Therefore, herbivore-induced plnt voltiles my be detectble nd relible indictors of herbivore presence nd identity. Plnt odours tht ttrct nturl enemies re clled herbivore-induced synomones (Vet & Dicke, 1992). Dmge by herbivores cn gretly increse the emission of plnt voltiles (Mtticci et l. 1994; Mumm et l., 2003; Bukovinszky et l., 2005) nd these plnt voltiles my be specific indictors of herbivore identity (Dicke et l., 1990b). Herbivory cn lso induce de novo production of compounds in mny plnt species, resulting in qulittive chnges in composition of the emitted blend (Turlings et l., 1990; Mores et l., 1998; Dicke, 1999). The relese of herbivore-induced plnt voltiles hs been shown to constitute n ctive response of the plnt, s is pprent from de novo production of voltile compounds tht re not relesed by intct or mechniclly dmged plnts (Dicke et l., 1990; Dicke et l., 1990b; Turlings et l., 1990). The min components of the voltile blend relesed from Brssic plnts re terpenoids nd green lef voltiles (Mtticci et l., 1994; Shiojiri et l., 2001). Terpenoids re mjor clss mong voltiles tht ttrct nturl enemies (Tkbyshi et l., 1994; Pichersky & Gershenzon, 2002). They re relsed in nlogous mounts in herbivore-dmged nd 4

9 Chpter I mechniclly-dmged cbbge plnts, s well s in undmged plnts (Mtticci et l., 1994; Shiojiri et l., 2001). The voltile frction of terpenoids predominntly consists of the hemiterpene soprene (C 5 ), monoterpenes (C 10 ) nd the sesquiterpenes (C 15 ) nd their derivtes such s homoterpenes (C 11 nd C 16 ). Monoterpenes nd sesquiterpenes re synthezised by the condenstion of two or three isoprene units, respectively (Cheng et l., 2007). Becuse of their physiochemicl properties, such s voltility, rectivity nd toxicity mny protective functions ginst biotic nd biotic fctors hve been documented for terpenoids (Holopinen, 2004). Terpenoids re involved in plnt-pollintor interctions nd hve importnt functions in plnt defense ginst herbivores (Pré & Tumlinson, 1999; Pichersky & Gershenzon, 2002; Keeling & Bohlmnn, 2006; Schie et l., 2006). The plnt-relesed C 6 ldehydes, lcohols nd esters re clled green lef voltiles, becuse they embody the typicl odour of undmged nd dmges leves. They re brekdown products formed by oxidtion of plnt lipids (Htnk, 1993). Plnts emit green lef voltiles during ging or when injury occurs (Visser & Avé, 1978; Htnk, 1993). There is drmtic increse of green lef voltiles in the hedspce of dmged cbbge plnts compred to undmged plnts, which my impct host serching of prsitoids (Mtticci et l., 1994; Smid et l., 2002). Isothiocyntes re voltile brekdown products of glucosinoltes which re found in Brssiccee (Rsk et l., 2000; Hopkins et l., 2009;). Glucosinoltes re metbolized to the toxic isothiocyntes nd other compounds by the enzym myrosinse which is stored in spezilised plnt cells. When the plnt tissue is dmged, e.g. by herbivores, the glucosinoltes stored in the vcuole come into contct with the enzyme myrosinse. Specilist herbivores on Brssiccee, re ble to detoxify, excrete or reduce the toxicity by behviourl dpttion (Hopkins et l., 2009). More thn 120 different glucosinoltes hve been identified so fr (Fhey et l., 2001). Nturl enemies use glucosinoltes nd their brekdown products s synomones to find their hosts within their host plnt. Butenyl-isothiocynte, which ws found to ttrct Phrdis morionellus is relesed in higher mounts from plnts infested by pollen beetle lrve thn from uninfested plnts (Jönsson & Anderson, 2008). The prsitoids T. obscurtor nd T. fulvipes nd Pltygster subuliformis were strongly ttrcted to yellow wter trps bited with 2-phenylethyl isothiocynte, component of the odour of the vegettive prts of oilseed rpe (Murchie et l., 1997; Ulber & Wedemeyer, 2006) 5

10 Chpter I Possible effects of insecticides on prsitoids Current pest control prctices re bsed on brod spectrum chemicl insecticides which lso hve neurotoxic side-effects to beneficil insects. Two or three insecticide tretments were performed to control the mjor pests ttcking oilseed rpe, the rpe stem weevil nd cbbge stem weevil t stem elongtion, the pollen beetle t bud stge nd the cbbge seed weevil nd the brssic pod midge t flowering (Alford et l., 2003; Menzler-Hokknen et l., 2006; Bürger & Gerowitt, 2009; Freier et l., 2009). Frequent pplictions of pyrethroid insecticides hve recently cused widespred resistnce of pollen beetles in mny Europen countries (Hnsen, 2003; Heimbch et l., 2006; Wegorek & Zmojsk, 2006). Further, insecticides my hrm nturl enemies of pests. The min period of ctivity of tersilochine prsitoids (except Tersilochus microgster) occurs in the lte bud stge up to the end of flowering of oilseed rpe (Johnen et l., 2006; Ulber et l., 2006). Adult prsitoids cn be ffected by insecticides in different wys: they cn be exposed to insecticide spry droplets (Jepson, 1989) or to the residues on the plnt folige when forging for hosts (Brown, 1989; Longley & Jepson, 1996) or feeding on contminted wter droplets nd nectr (Lngley & Strk, 1996). Prsitoids my be ffected by incresed direct mortlity or by sublethl effects on their physiology nd behviour. Sublethl effects of insecticides re defined s effects (pyhsiologicl or behviourl) on individuls tht survive exposure to pesticides (Desneux et l., 2007). Sublethl effects of insecticides my ffect fecunditiy, longevity nd behviour through neurotoxic ctivity. Behviourl effects of insecticides on prsitoids my be knock-down, uncoordinted movements (Desneux et l., 2004), more frequent clening of their bdomen nd legs nd repellent or irritnt effect of the pesticide (Wiles & Jepson, 1994; Longley & Jepson, 1996; Desneux et l., 2007). Further, the oviposition behviour of prsitoids my be disrupted by msking of the ttrctive host plnt odours, thereby preventing prsitoid femles from recognising the host plnt nd the host (Thiery & Visser, 1986; Nottinghm et l., 1991), or the insecticide odour could hve direct repellent effect on the insects (Jiu & Wge, 1990; Longley & Jepson, 1996; Trn et l., 2004). The phid prsitoid D. rpe spend less time forging on Brussels sprout plnts treted with the insecticides pirimicrb, permethrin nd mlthion compred to untreted plnts (Jiu & Wge, 1990; Umoru et l., 1996). 6

11 Chpter I Strtegies for conservtion of prsitoid popultions in integrted pest mngement systems According to threshold vlues of the pest popultions, IPM mngement systems on winter oilseed rpe will require chemicl pesticides. The effect of insecticides on prsitoid popultions my be ffected by the mode of ction, dosge, persistency nd temporl nd sptil ppliction (Jepson, 1989; Poehling, 1989; Alford et l., 1992). One wy to protect nturl enemies is to optimize the timing of insecticide ppliction. At erly tretments for control of stem weevils nd pollen beetles it is necessry to observe only the cbbge stem fle beetle prsitoid T. microgster. Most prsitoid species re ctive on oilseed during the lte bud stge or flowering. The use of phenologicl models to find spry windows tht llow insecticide tretments consistent with good pest control nd minimized effects on prsitoids could be utilized in IPM systems. Trgeting of insecticide ppliction to the crop with regrd to pest incidence nd prsitoid phenology on the crop hs potentil to minimize mortlity of prsitoids (Johnen et l., 2006). Selective insecticides might hve potentil to void hrmful effects of insecticides on prsitoids in winter oilseed rpe (Iwt et l., 1985; Brunner et l., 2001). Differentil toxicity of vrious pyrethroids nd reltively low toxicity of tu-fluvlinte to prsitoids in oilseed rpe (Klukowski et l., 2006; Jckowski et l., 2009) nd other crops hve been reported (Moreby et l., 2001). While pyrethroids re known to hve brod-spectrum ctivity (Csid et l., 1983), systemic insecticides, e.g. neonicotinoids, nd their metbolites re climed to be firly sfe for beneficil insects, becuse direct contmintion only occurs when the insects feed on the plnt tissue (Stpel et l., 2000). Knowledge of the reltive toxicities of different insecticides to prsitoids would id selection of ppropite products which could help to conserve nturl enemies nd could be used in IPM systems. Another wy for incresing the selectivity of insecticides my be using reduced ppliction rtes of insecticides. Applying insecticides t lower rtes of ctive ingredients per unit re my protect nturl enemies, wheres pest mortlity remins t high level (Ripper, 1956; Poehling, 1989; Longley et l., 1997; Booth et l., 2007). Most studies on the effect of reduced field dose rte on pests nd their nturl enemies hve been conducted with phids. It hs been proved in vrious studies tht reduced rtes of insecticides cn provide sufficient control of phids (Ripper 1956; Poehling, 1989; Achempong & Strk, 2004; Booth et l., 7

12 Chpter I 2007), whers nturl enemies were protected (Longley et l., 1997; Niehoff & Poehling, 1995). Sptil trgeting of insecticides to res of high pest density could be scope for reducing insecticide side-effects nd conserving prsitoid popultions. Pests re often distributed irregulrly within the crop. Mjor pests re edge-distributed during their immigrtion into the crop. This hs been observed with cbbge stem weevil (Free & Willims, 1979; Klukowski et l., 2006), pollen beetle (Free & Willims, 1979), cbbge stem weevil (Free & Willims, 1979; Murchie et l., 1999) nd brssic pod midge (Free & Willims, 1979; Ferguson et l., 2004). Appliction of insecticides only to crop edges during the immigrtion period of the pests could kill the pests while protecting the prsitoids in the centrl unspryed re of the crop (Brri et l. 2005; Cook et l., 2006). This could be mplified by using trps crops: this so clled push-pull strtegy is focused on the mnipultion of the distribution of the pests on the crop. In oilseed rpe, turnip rpe (Brssic rp L.) cn be used s the trp crop to pull the pests wy from the min oilseed rpe crop (Cook et l., 2006; Cook et l., 2007). Field experiments demonstrted tht turnip rpe crop borders reduce the bundnce of cbbge stem fle beetle (Brri et l., 2005) nd pollen beetle (Cook et l., 2006) in the oilseed rpe min crop nd reduce the need for insecticides. References ACHEAMPONG, S. & STARK, J. (2004). Cn reduced rtes of pymetrozine nd nturl enemies control the cbbge phid, Brevicoryne brssice (Homopter: Aphidide), on broccoli? Interntionl Journl of Pest Mngement 50, ALFORD, D. V., MURCHIE, A. & WILLIAMS, I. H. (1992). Observtion on the impct of stndrd insecticide tretments on Trichomlus perfectus, prsitoid of seed weevil on winter oilseed rpe in the UK. IOBC/wprs Bulletin 18, ALFORD, D. V., NILSSON, C. & ULBER, B. (2003). Insect pests of oilseed rpe crops. Chpter 2 In: D.V. Alford (ed) Biocontrol of Oilseed Rpe Pests. Blckwell, Oxford, UK, BARARI, H., COOK, S. M. & WILLIAMS, I. H. (2005). Effect of turnip rpe (Brssic rp) trp crop on stem-mining pests nd their prsitoids in winter oilseed rpe (Brssic npus). Biologicl Control 50, BOOTH, L. H., WRATTEN, S. D. & P.KEHRLI. (2007). Effects of reduced rtes of two insecticides on enzyme ctivity nd mortlity of n phid nd its lcewing predtor. Journl of Economic Entomology 100, BROSCHEWITZ, B. (1985). Untersuchungen zur Biologie und Schdwirkung des Gefleckten Kohltriebrüsslers (Ceutorhynchus qudridens Pnzer) m Winterrps (Brssic npus L. vr. oleifer Metzg.). PhD-thesis, Wilhelm-Pieck-Universität, Rostock. 8

13 Chpter I BROSCHEWITZ, B. & DAEBLER, F. (1987). Beitrg zur Biologie und Schdwirkung des Gefleckten Kohltriebrüsslers (Ceutorhynchus qudridens Pnz) n Winterrps. Nchrichtenbltt für den Pflnzenschutzdienst in der DDR 41, BROWN, K. C. (1989). The design of experiments to ssess the effects of pesticides on beneficil rthropods in orchrds: Repliction versus plot size. In: Jepson P.C. (Ed.), Pesticide nd Non-trget Invertebrtes. Intercept, Wimborne., BRUNNER, J. F., DUNLEY, J. E., DOERR, M. D. & BEERS, E. H. (2001). Effect of Pesticides on Colpoclypeus florus (Hymenopter: Eulophide) nd Trichogrmm pltneri (Hymenopter: Trichogrmmtide), Prsitoids of Lefrollers in Wshington. Journl of Economic Entomology 94, BÜCHI, R. & ROOS-HUMBEL, S. (1991). Nützlinge reduzieren die Zhl der Rpsschädlinge. Lndwirtschft Schweiz 4, BÜRGER, J. & GEROWITT, B. (2009). Anwendungsmuster von Pflnzenschutzmitteln in Winterweizen und Winterrps. Gesunde Pflnze 61, BUKOVINSZKY, T., GOLS, R., POSTHUMUS, M. A., VET, L. E. M. & LENTEREN, J. C. V. (2005). Vrition in plnt voltiles nd ttrction of the prsitoid Didegm semiclusum (Hellén). Journl of Chemicl Ecology 31, CASIDA, J. E., GAMMON, D. W., GLICKMAN, A. H. & LAWRENCE, L. J. (1983). Mechnism of Selective Action of Pyrethroid Insecticides. Annul Review Phrmcology Toxicology 23, CHENG, A., LOU, Y., MAO, Y., LU, S., WANG, L. & CHEN, X. (2007). Plnt Terpenoids: Biosynthesis nd Ecologicl Functions. Journl of Integrtive Plnt Biology 49, COOK, S. M., KHAN, Z. R. & PICKETT, J. A. (2007). The use of "push-pull" strtegies in integrted pest mngement. Annul Review Entomology 52, COOK, S. M., SMART, L. E., MARTIN, J. L., MURRAY, D. A., WATTS, N. P. & WILLIAMS, I. H. (2006). Exploittion of host plnt preferences in pest mngement strtegies for oilseed rpe (Brssic npus). Entomologi Experimentlis et Applict 119, DEBOUZIE, D. & BALLANGER, Y. (1993). Dynmics of Ceutorhynchus npi popultion in winter rpe fields. Act Oecologic 14, DESNEUX, N., DECOURTYE, A. & DELPUECH, J.-M. (2007). The sublethl effects of pesticides on beneficil rthropods. Annul Reviews Entomology 52, DESNEUX, N., WAJNBERG, E., FAUVERGUE, X., PRIVET, S. & KAISER, L. (2004). Oviposition behviour nd ptch-time lloction in two phid prsitoids exposed to deltmthrin residues. Entomologi Experimentlis et Applict 112, DICKE, M. (1999). Are herbivore-induced plnt voltiles relible indictors of herbivore identity to forging crnivorous rthropods? Entomologi Experimentlis et Applict 91, DICKE, M., BEEK, T. A. V., POSTHUMUS, M. A., DOM, N. B., BOKHOVEN, H. V. & GROOT, A. D. (1990). Isoltion nd identifiction of voltile kiromones tht ffects crine predtor-prey interctions. Journl of Chemicl Ecology 16, DICKE, M., SABELIS, M. W., J.TAKABAYASHI, BRUIN, J. & POSTHUMUS, M. A. (1990b). Plnt strtegies of mnipulting predtor-prey interctions through llelochemicls: prospects for ppliction in pest control. Journl of Chemicl Ecology 16, FAHEY, J. W., ZALCMANN, A. T. & TALALAY, P. (2001). The chemicl diversity nd distribution of glucosinoltes nd isothiocyntes mong plnts. Phytochemistry 56, FAO. (2007). FERGUSON, A. W., CAMPBELL, J. M., WATTS, D. J., SCHMIDT, J. E. U. & WILLIAMS, I. H. (2004). Phenology nd sptil distribution of Dsineur brssice nd its prsitoids 9

14 Chpter I in crop of winter oilseed rpe: implictions for integrted pest mngement. IOBC/wprs Bulletin 27, FERGUSON, A. W., KLUKOWSKI, Z., WALCZAK, B., CLARK, S. J., MUGGLESTONE, M. A., PERRY, J. N. & WILLIAMS, I. H. (2003). Sptil distribution of pests insects in oilseed rpe: implictions for integrted pest mngement. Agriculturl, Ecosystems nd Environment 95, FREIER, B., PALUTT, B., JAHN, M., SELLMANN, J., GUTSCHE, V., ZORNBACH, W. & MOLL, E. (2009). Netz Vergleichsbetriebe Pflnzenschutz Jhresbericht Berichte us dem Julius Kühn-Institut 149. FLINT, M. L. & BOSCH, R. V. D. (1981). Introduction to Integrted Pest Mngement. Plenum, New York, USA. FREE, J. B. & WILLIAMS, I. H. (1979). The distribution of insect pests on crops of oilseed rpe (Brssic npus L.) nd the dmge they cuse. Journl of Agriculturl Science 93, HANSEN, L. M. (2003). Insecticide-resistnt pollen beetles (Meligethes eneus F) found in Dnish oilseed rpe (Brssic npus L) fields. Pest Mngement Science 59, HATANAKA, A. (1993). The biogenertion of green odour by green leves. Phytochemistry 34, HEIMBACH, U., MÜLLER, A. & THIEME, T. (2006). First steps to nlyse pyrethroid resistnce of different oilseed rpe pests in Germny: An extended bstrct. IOBC/wprs Bulletin 29, HOKKANEN, H., HUSBERG, G.-B. & SÖDERBLOM, M. (1988). Nturl enemy conservtion for the integrted control of the rpe blossom beetle Meligethes eneus F. Annles Agriculture Fennie 27, HOKKANEN, H. M. T. (1993). Overwintering survivl nd spring emergence in Meligethes eneus: effects of body weight, crowding, nd soil tretment with Beuveri bssin. Entomologi Experimentlis et Applict 67, HOLOPAINEN, J. K. (2004). Multiple functions of inducible plnt voltiles. Trends in Plnt Science 9, HOPKINS, R. J., DAM, N. M. V. & LOON, J. J. A. V. (2009). Role of Glucosinoltes in insectplnt reltionships nd multitrophic interctions. Annul Review Entomology 54, IWATA, Y., MACCONNELL, J. G., FLOR, J. E., PUTTER, I. & DINOFF, T. M. (1985). Residues of Avermectin B 1 on nd in Citrus Fruits nd Folige. Journl of Agriculturl Food Chemistry 33, JACKOWSKI, J., KLUKOWSKI, Z. & IRZYKOWICZ, M. (2009). The effect of tu-fluvlinte nd lmbd-cyhlothrin on two prsitic species of Phrdis spp. (Hymenopter, Ichneumonide, Tersilochine). Pestycydy/Pesticides , JEPSON, P. C. (1989). The temporl nd sptil dynmics of pesticide side-effects on nontrget invertebrtes. In: Jepson P.C. (Ed.), Pesticide nd Non-trget Invertebrtes. Intercept, Wimborne., JIU, G. D. & WAAGE, J. K. (1990). The effect of insecticides on the distribution of forcing prsitoids, Dieretiell rpe (Hym.: Brconide) on plnts. Entomophg 35, JOHNEN, A., WILLIAMS, I. H., FERGUSON, A. W., BÜCHS, W., KLUKOWSKI, Z., LUIK, A., NILSSON, C. & ULBER, B. (2006). MASTER: construction of phenologicl models of key prsitoids in Europe nd prospects for spry windows comptible with their conservtion in winter oilseed rpe. CD-Rom Proceedings of the Interntionl Symposium "Integrted Pest Mngement in Oilseed Rpe Pests", Goettingen, Germny, 3-5 April

15 Chpter I JÖNSSON, M. & ANDERSON, P. (2008). Emission of oilseed rpe voltiles fter pollen beetle infesttion; behviourl nd electrophysiologicl responses in the prsitoid Phrdis morionellus. Chemoecology 17, JOURDHEUIL, P. (1960). Influence de quelque fcteurs écologique sue les fluctutions de popultion d une biocé nose prsitire. Ann. Epiph. 11, KAISER, L. & CARDÉ, R. T. (1992). In flight orienttion to voltiles from the host-plnt complex in Cotesi rubecul (Hym.: Brconide): Incresed sensitivity through olfctory experience. Physiologicl Entomology 17, KEELING, C. I. & BOHLMANN, J. (2006). Genes, enzymes nd chemicls of terpenoid diversity in the constitutive nd induced defence of conifers ginst insects nd pthogens. New Phytologist 170, KLINGENBERG, A. (1991). Untersuchungen zur Abundnzerfssung und zu ntürlichen Gegenspielern von Ceutorhynchus pllidctylus (Mrsh) und Ceutorhynchus npi (Gyll.). Diplomrbeit, Universität Göttingen. KLINGENBERG, A. & ULBER, B. (1994). Untersuchungen zum Auftreten der Tersilochine (Hymenopter: Ichneumonide) ls Lrvlprsitoiden einiger Rpsschädlinge im Rum Göttingen 1990 und 1991 und zu deren Schlupfbundnz nch unterschiedlicher Bodenberbeitung. Journl of Applied Entomology 117, KLUKOWSKI, Z., TWARDOWSKI, J. & IRZYKOWICZ, M. (2006). Subsequent effect of tufluvlinte (Mverik) nd lmbd-cyhlothrin (Krte) pyrethroids on the ctivity of crbid beetles (Coleopter: Crbide) in winter oilseed rpe. IOBC/wprs Bulletin 29, LAMB, R. J. (1989). Entomology of oilseed Brssic crops. Annul Review of Entomology 43, LANGLEY, M. & STARK, J. D. (1996). Anlyticl Techniques for quntifying direct, residul nd orl exposure of n insect prsitoid to n Orgnophosphte Insecticide. Bulletin of Environmentl Contmintion Toxicology 57, LEHMANN, W. (1969). Beiträge zur Prsitenfun in Rpsbeständen. Bericht über die 10. Wnderversmmlung Deutscher Entomologen, Dresden 1965, Deutsche Akdemie der Lndwirtschft zu Berlin, Tgungsbericht 80, Teil III, LONGLEY, M. & JEPSON, P. C. (1996). The influence of insecticide residues on primry prsitoids nd hyperprsitoid forging behviour in the lbortory. Entomologi Experimentlis et Applict 81, LONGLEY, M., JEPSON, P. C., IZQUIERDO, J. & SOTHERTON, N. (1997). Temporl nd sptil chnges in phid nd prsitoid popultions following pplictions of deltmethrin in winter whet. Entomologi Experimentlis et Applict 83, MATTIACCI, L., DICKE, M. & POSTHUMUS, M. A. (1994). Induction of prsitoid ttrcting synomones in brussels sprouts plnts by feeding of Pieris brssice lrve: Role of mechnicl dmge nd herbivore elicitor. Journl of Chemicl Ecology 20, MORAES, C. M. D., LEWIS, W. J., PARÉ, P. W., ALBORN, H. T. & TUMLINSON, J. H. (1998). Herbivore-infested plnts selectively ttrct prsitoids. Nture 393, MOREBY, S. J., SOUTHWAY, S., BARKER, A. & HOLLAND, J. M. (2001). A comprison of the effect of new nd estblished insecticides on nontrget invertebrtes of winter whet fields. Environmentl Toxicology Chemistry 20, MUMM, R., SCHRANK, K., WEGENER, R., SCHULZ, S. & HILKER, M. (2003). Chemicl nlysis of voltiles emitted by Pinus sylvestris fter induction by insect oviposition. Journl of Chemicl Ecology 29, MURCHIE, A. K., SMART, L. E. & WILLIAMS, I. H. (1997). Responses of Dsineur brssice nd its prsitoids Pltygster subuliformis nd Omphle clypelis to field trps bited with orgnic isothiocyntes. Journl of Chemicl Ecology 23,

16 Chpter I MURCHIE, A. K., WILLIAMS, I. H. & PERRY, J. N. (1999). Edge distribution of Ceutorhynchus ssimilis nd its prsitoid Trichomlus perfectus in crop of winter oilseed rpe (Brssic npus). BioControl 44, NIEHOFF, B. & POEHLING, H. M. (1995). Popultion dynmics of phids nd syrphid lrve in winter whet treted with different rtes of pirimicrb. Agriculturl, Ecosystems nd Environment 52, NILSSON, C. (1988). The pollen beetle (Meligethes eneus F.) in winter nd spring rpe t Alnrp III. Mortlity fctors. Växtskyddsnotiser 52, NILSSON, C. (2003). Prsitoids of pollen beetles. In: D.V. Alford (ed) Biocontrol of Oilseed Rpe Pests. Blckwell Publishing, Oxford, UK, NILSSON, C. & ANDREASSON, B. (1987). Prsitoids nd predtors ttcking pollen beetles (Meligethes eneus F.) in spring nd winter rpe in southern Sweden. IOBC/wprs Bulletin 4, NISSEN, U. (1997). Ökologische Studien zum Auftreten von Schdinsekten und ihren Prsitoiden n Winterrps norddeutscher Anbugebiete. Disserttion, Universität Kiel. NISSEN, U. (1999). Ntürliche Gegenspieler von Rpsschädlingen. Rps 17, NITZSCHE, O. (1998). Auftreten und Effizienz von Prsitoiden ls ntürliche Gegenspieler von Schdinsekten im Winterrps unter besonderer Berücksichtigung unterschiedlicher Bodenberbeitungsmßnhmen nch Winterrps. PhD thesis, Georg-August-University of Goettingen, Germny. NOTTINGHAM, S. F., HARDIE, J., DAWSON, G. W., HICK, A. J., PICKETT, J. A., WADHAMS, L. J. & WOODCOCK, C. M. (1991). Behviorl nd electrophysiologicl responses of phids to host nd nonhost plnt voltiles. Journl of Chemicl Ecology 17, OSBORN, P. (1960). Observtion of the nturl enemies of Meligethes eneus (F.) nd M. viridescence (F.) (Coleopter: Nitidulide). Prsitology 50, PARÉ, P. W. & TUMLINSON, J. H. (1997). Induced synthesis of plnt voltiles. Nture 385, PARÉ, P. W. & TUMLINSON, J. H. (1999). Plnt voltiles s defense ginst herbivores. Plnt Physiology 121, PAUL, V. H. (2003). Rps-Krnkheiten, Schädlinge, Schdpflnzen. Th. Mnn Verlg, Gelsenkirchen-Buer. PICHERSKY, E. & GERSHENZON, J. (2002). The formtion nd function of plnt voltiles: perfumes for pollintor ttrction nd defense. Current Opinion in Plnt Biology 5, POEHLING, H. M. (1989). Selective ppliction strtegies for insecticides in griculturl crops. In: Jepson P.C. (Ed.), Pesticide nd Non-trget Invertebrtes. Intercept, Wimborne., POTTING, R. P. J., POPPY, G. M. & SCHULER, T. H. (1999). The role of voltile from cruciferous plnts nd pre-flight experience in the forging bhviour of the specilist prsitoid Cotesi plutelle. Entomolgi Experimentlis et Applict 93, RASK, L., ANDRÉASSON, E., EKBOM, B., ERIKSSON, S., PONTOPPIDAN, B. & MEIJER, J. (2000). Myrosinse: gene fmily evolution nd herbivore defense in Brssiccee. Plnt Moleculr Biology 42, RIPPER, W. E. (1956). Effect of pesticides on blnce of rthropod popultions. Annul Reviews Entomology 1, SCHIE, C. C. N. V., HARING, M. A. & SCHURINK, R. C. (2006). Regultion of terpenoids nd benzenoids production in flowers. Current Opinion in Plnt Biology 9,

17 Chpter I SCHMUTTERER, H. (1956). Zur Lebensweise und Bekämpfung des Großen Rpsstängelrüsslers Ceutorhynchus npi (Gyll.). Zeitschrift Angewndten Entomologie 39, SEDIVY, J. (1983). Tersilochine s prsitoids of insect pests of winter rpe (Hymenopter: Ichneumonide). Cont. Am. Ent. Inst. 20, SHIOJIRI, K., TAKABAYASHI, J., YANO, S. & TAKAFUJI, A. (2001). Infochemiclly medited tritrophic interction webs on cbbge plnts. Popultion Ecology 43, SMID, H. M., LOON, J. J. A. V., M.A.POSTHUMUS & VET, L. E. M. (2002). GC-EAG-nlysis of voltiles from Brussels sprouts plnts dmged by two species of Pieris cterpillrs: olfctory receptive rnge of specilist nd generlist prsitoid wsp species. Chemoecology 12, STAPEL, J. O., CORTESERO, A. M. & LEWIS, W. J. (2000). Disruptive Sublethl Effects of Insecticides on Biologicl Control: Altered Forging Ability nd Life Spn of Prsitoid fter Feeding on Extrflorl Nectr of Cotton Treted with Systemic Insecticides. Biologicl Control 17, TAKABAYASHI, J. & DICKE, M. (1996). Plnt-crnivore mutulism through herbivore-induced crnivore ttrctnts. Trends in plnt science 1, TAKABAYASHI, J., DICKE, M. & POSTHUMUS, M. A. (1994). Voltile herbivore-induced terpenoids in plnt-mite interctions: Vrition cused by biotic nd biotic fctors. Journl of Chemicl Ecology 20, THIERY, D. & VISSER, J. H. (1986). Msking of plnt odour in the olfctory orienttion of the Colordo potto beetle. Entomologi Experimentlis et Applict 41, TRAN, D. H., TAKAGI, M. & TAKASU, K. (2004). Effects of selective insecticides on host serching nd oviposition behvior of Neochrysochris formos (Westwood) (Hymenopter: Eulophide), lrvl prsitoid of the Americn serpentine lefminer. Applied Entomology Zoology 39, TURLINGS, T. C. J., TUMLINSON, J. H., HEATH, R. R., PROVEAUX, A. T. & DOOLITTLE, R. E. (1991). Isoltion nd identifiction of llelochemicls tht ttrct the lrvl prsitoid, Cotesi mrginiventris (Cresson), to the microhbitt of one of its hosts. Journl of Chemicl Ecology 17, TURLINGS, T. C. J., TUMLINSON, J. H. & LEWIS, W. J. (1990). Exploittion of herbivoreinduced plnt odors by host-seeking prsitic wsps. Science 250, UFOP. (2009). ULBER, B. (2003). Prsitoids of Ceutorhynchid stem weevils. In: D.V. Alford (ed) Biocontrol of Oilseed Rpe Pests. Blckwell Publishing, Oxford, UK, ULBER, B., NITZSCHE, O. & WEDEMEYER, R. (2006). Phenology of tersilochine prsitoids in Germny nd prospects for spry windows comptible with their conservtion. CD- Rom Proceedings of the Interntionl Symposium "Integrted Pest Mngement in Oilseed Rpe Pests", Goettingen, Germny, 3-5 April ULBER, B. & WEDEMEYER, R. (2006). Responses of Tersilochus microgster nd Tersilochus obscurtor (Hymenopter: Ichneumonide) to voltile 2-phenylethyl-isothiocynte. CD-Rom Proceedings of the Interntionl Symposium "Integrted Pest Mngement in Oilseed Rpe Pests", Goettingen, Germny, 3-5 April UMORU, P. A., POWELL, W. & CLARK, S. J. (1996). Effect of pirimicrb on the forging behviour of Dieretiell rpe (Hymenopter: Brconide) on host-free nd infested oilseed rpe plnts. Bulletin of Entomologicl Reserch 86, VET, L. E. M. & DICKE, M. (1992). Ecology of infochemicl use by nturl enemies in tritrophic context. Annul Review Entomology 37, VET, L. E. M., WÄCKERS, F. L. & DICKE, M. (1991). How to hunt for hiding hosts: The relibility-detectbility problem in forging prsitoids. Netherlnds Journl of Zoology 41,

18 Chpter I VINSON, S. B. (1998). The generl host selection behviour of prsitoid Hymenoptern nd comprison of initil strtegies utilized by lrvphgous nd oophgous species. Biologicl Control 11, VISSER, J. H. & AVÉ, D. A. (1978). Generl green lef voltiles in the olfctory orienttion of the colordo beetle, Leptinotrs decemlinet. Entomologi Experimentlis et Applict 24, WEGOREK, P. & ZAMOJSKA, J. (2006). Resistnce of pollen beetle (Meligethes eneus F.) to pyrethroids, chloronicotinyls nd orgnophosphorous insecticides in Polnd. IOBC/wprs Bulletin 29, WILES, J. A. & JEPSON, P. C. (1994). Sub-lethl effects of deltmethrin residues on the within-crop behviour nd distribution of Coccinell septempunctt. Entomologi Experimentlis et Applict 72, WILLIAMS, I. H., FREARSON, D. J. T., BARARI, H. & MCCARTNEY, A. (2007). First field evidence tht prsitoids use upwind nemotxis for host-hbitt loction. Entomologi Experimentlis et Applict 123, WINFIELD, A. L. (1963). A study on the effects of insecticides on prsites of lrve of blossom beetles (Meligehtes eneus F., Coleopter: Nitidulide). Entomologi Experimentlis et Applict 6, WINFIELD, A. L. (1992). Mngement of oilseed rpe pests in Europe. Agriculturl Zoology Reviews 5,

19 Chpter II Role of voltiles emitted from the host-plnt complex in host loction by the cbbge stem borer prsitoid Tersilochus obscurtor on winter oilseed rpe Abstrct The cbbge stem weevil, Ceutorhynchus pllidctylus, is n importnt pest on crops of winter oilseed rpe. Dmge to plnts is cused by lrvl feeding within petioles nd stems. Weevil popultions re under prtil nturl control by the key lrvl prsitoid Tersilochus obscurtor. In this study chemicl stimuli which re used by femle T. obscurtor for finding their endophgous host lrve were investigted. In lbortory experiments, the role of voltiles emitted from uninfested plnts nd from plnts infested by lrve of C. pllidctylus ws studied in behviourl nd electrophysiologicl (EAG-GC/MS) biossys, using femle T. obscurtor s test insects. Nive nd experienced femles were used in dul-choice tests nd Y-olfctometer biossys. Prior experience with host-plnt cues significntly incresed the probbility of host finding. In olfctometer tests, prsitoids responded to voltile cues elicited by plnts dmged by stem weevils. Odour of host lrve did not ttrct the wsps. Six of the 19 identified voltiles were relesed in significntly higher mounts from infested plnts thn from uninfested plnts. Further, eight compounds were only relesed from infested plnts nd one compound only from uninfested plnts. In EAG biossys, femle T. obscurtor strongly responded to voltile compounds relesed from infested plnts, especilly Nonnl nd α-frnesene, nd to 1-Hexnol, 2-ethyl, nd Terpinen-4-ol relesed from uninfested plnts. Introduction The cbbge stem weevil, Ceutorhynchus pllidctylus (Mrsh.) (Col., Curculionide), is one of the most dmging stem-boring pests in winter oilseed rpe (Brssic npus L. vr. oleifer Metzg.) in Europe (Alford et l., 2003). Femles deposit their egg btches into petioles of oilseed rpe plnts in April/My. Dmge is cused by lrvl feeding within petioles nd lter in the min stem. In Germny yield losses of 20-30% hve been reported (Broschewitz et l., 1993). The lrve of C. pllidctylus re prsitized by the univoltine endoprsitoid Tersilochus obscurtor Aubert (Hymenopter: Ichneumonide) (Ulber 2003; Ulber & Nitzsche, 2006). Femle prsitoids ly their eggs singly into the host lrve while these re mining within petioles. Prsitoid lrve htch within hosts, but develop to second nd third instr lrve not before the full-grown host lrve hs migrted to soil for puption 15

20 Chpter II (koinobiont). The dult wsps dipuse inside their pupl coocon in the soil until emerge in the following spring (Ulber, 2003). Forging insect prsitoids re known to respond to vriety of stimuli, including chemicl, visul, vibrtionl, nd tctile cues (Allen et l., 1999; Meyhöfer & Css, 1999; Wäckers & Lewis, 1999; Jönsson et l. 2005). Olfction is often considered to provide the most importnt evidence for the presence of endophgous host lrve (Vet & Dicke, 1992; Potting et l. 1995; Dicke 1999). Prsitoids hve been found to respond to plnt odours more esily thn to host derived stimuli (Vet et l. 1991). Vrious plnt voltiles re relesed specificlly fter herbivory. These compounds cn be relible indictors for host presence nd ttrct prsitoids (Turlings et l. 1990). As these chemicls re fvourble to the plnt, they re clled herbivore-induced synomones (Vet & Dicke, 1992). Little is known bout the behviourl ecology of T. obscurtor nd in prticulr bout fctors ffecting host loction. The im of this study ws to investigte, in lbortory biossys, the olfctoric stimuli which re used by T. obscurtor for encounter of host lrve. A coupled electrophysiology-gschromtogrphy pproch ws used to sub-divide the complex plnt voltile blends into individul components, nd to identify which individul voltile elicits olfctory ctivity in prsitoid femles. Mteril nd Methods Lbortory experiments Test insects To obtin lrge number of femle T. obscurtor of stndrdized ge nd of similr physiologicl condition for the biossys, lrve of C. pllidctylus were collected from min stems of unspryed oilseed rpe plnts in the field. Third instr lrve were relesed on slices of kohlrbi in plstic boxes nd rered until mturtion. For puption, the full-grown lrve were trnsferred to plstic boxes (500 ml) contining moistened soil substrte (cly-lomy soil mixed with snd). The boxes were stored under lbortory conditions (16hL : 8hD, 22 C). Adult cbbge stem weevils emerging from the soil fter dys were trnsferred to plstic boxes nd supplied with leves of oilseed rpe. To enble n obligte prereproductive dipuse, the weevils were stored under simulted winter conditions in cooling room t chnging temperture nd light regime (Tble 1). By using this method, totl of 33 to 44% of lrve ws successfully rered up to the dult stge. 16

21 Chpter II Tble 1: Temperture nd light regime pplied to dipusing dults of C. pllidctylus Period temperture light regime dys C (h L : h D) : : : : : lbortory 18 : 6 For rering dult T. obscurtor in the lbortory, prsitoids cocoons were collected from the soil used for puption of weevils by sieving (mesh size 2 mm) nd stored in petri dishes lined with moisture filter pper. As the prsitoid requires n obligte pre-productive dipuse, the cocoons were lso kept in cooling room under simulted winter conditions t chnging temperture nd light regime (Tbel 2). In totl, 28-40% of field collected C. pllidctylus lrve were prsitized. Tble 2: Temperture nd light conditions pplied to dipusing T. obscurtor Period temperture light regime dys C (h L : h D) : : : : : 6 Emergence of dult prsitoids ws initited by trnsferring the cocoons to lbortory conditions. Before trnsferring to the experiments, femles nd mles were stored for 5 8 dys in cge under lbortory conditions. They were supplied with oilseed rpe flowers nd wter. In the two-choice tests femles with oviposition experience nd unexperienced femles were used. The effect of oviposition experience on the response of prsitoids to voltile cues emitted from the host-plnt complex ws investigted by using the following methodology. One group consisted of nive femles which hd no prior contct with infested plnt mteril before used in the biossy. The other group consisted of experienced femles which were relesed for 1h on leves infested by C. pllidctylus lrve nd llowed to oviposit 2h prior to the biossy. 17

22 Chpter II Collection of dult prsitoids in the field For the GC/MS-EAG nlyses, field collected prsitoids were used. They were cptured in mlise trps which were exposed in insecticide-untreted fields of oilseed rpe crop ner Göttingen. The trps were emptied twice dy. Femle prsitoids were stored for 48 hours in the lbortory nd supplied with oilseed rpe flowers nd wter before the experiment strted. Artificil infesttion of plnts by C. pllidctylus Plnts infested by lrve of C. pllidctylus were needed in the behviourl nd electrophysiologicl biossys with T. obscurtor. Oilseed rpe plnts (Brssic npus, cultivr Westr) were grown under greenhouse conditions. For rtificil infesttion by C. pllidctylus two plnts in the 6-true-lef stge were exposed to three femles nd one mle of C. pllidctylus within n insect cge (BugDorm-2, Meg View Science nd Eduction Services Co., Tichung, TW) mesuring 60cm x 60cm x 60cm. Following n oviposition time of 24h, plnts were removed nd trnsferred to climte chmber (20 C, 16 L : 8 D, 4.000lux). Infested plnts were used in the experiments 7 21 dys fter oviposition, when lrve were in the second to third instr. Dul-choice test A dul-choice experiment ws designed to study the behviour of nive nd experienced T. obscurtor femles, when given choice between n uninfested lef nd lef infested by C. pllidctylus lrve. In previous experiments in windtunnel, femles of T. obscurtor did not respond when exposed to different wind intensities nd different light sources. Better results could be obtined by offering infested leves in smll experimentl ren to prsitoid femles. In this experiment leves of oilseed rpe (Brssic npus, cultivr Westr), which hd been infested with lrve of C. pllidctylus s describes bove were used. An infested nd n uninfested lef were exposed 10 cm prt in perspex cge (30 cm x 20 cm), with two wlls covered with guze for ir exchnge. The bse of the petioles ws seled with wet cotton wool. The experiment ws performed in room without dylight. As prsitoids re highly ttrcted by the light, the light source ws set up below the experimentl cge. Five to eight dys old femles of T. obscurtor were relesed individully in the center of the experimentl cge. The totl residence time on the leves ws recorded within 5 min observtion time. In ddition, the first choice nd the ovipositor probes were ssessed. 18

23 Chpter II Y- Olfctometer tests The response of femle T. obscurtor to olfctory stimuli involved in host microhbitt loction ws studied by using Y-olfctometer. Five different sources of odour were tested: (1) infested lef with host lrve vs. uninfested lef; (2) infested lef with host lrve vs. infested lef without host lrve; (3) infested lef without host lrve vs. uninfested lef; (4) host lrve vs. clen ir; (5) uninfested lef vs. clen ir. In the Y- olfctometer, the insects could select between two irstrems crrying odours of the different tretments. It consisted of Y-shped perspex tube of 27 mm inner dimeter, with rms of 120 mm length connected t 40 ngle to the centrl tube. As T. obscurtor is strongly ttrcted by light, the light source ws positioned t the bck of the two rms of the olfctometer, thereby stimulting insect movement to this direction. The ir ws pumped into gs-wsh bottle filled with ctive chrcol nd therefter divided into two lines of Teflon tubing. The ir flow ws djusted vi two flow meters to 70 cm³/ sec. The ir ws piped into bottles with distilled wter. The tubes were connected to two bottles contining the odour sources. Groups of five femles of T. obscurtor were relesed into the centrl tube of 15 cm length nd llowed to choose one of the rms. Femles tht did not mke choice within 10 minutes were not included in the nlyses. The first choice for one of the two odour sources ws recorded when femles moved into the respective rm of the olfctometer. To void ny symmetricl bis in the setup, the odour sources were chnged fter testing 10 prsitoids. For ech combintion, 30 femles were tested. Collection of voltiles emitted from plnts Potted oilseed rpe plnts (cultivr Westr), s described bove, were used to collect the odour blend emitted from uninfested nd infested plnts. A totl of 25 uninfested nd 68 infested individul oilseed rpe plnts were tested. Plnts infested by C. pllidctylus lrve were enclosed within n ethylen-tetrflorethylen (ETFE) bg (70 cm x 40 cm x 40 cm) in the greenhouse under dylight conditions, from 10 m to 1 pm. The bgs were mounted on lbortory pedest. The opening of the bg ws closed round the stem bse by mounting its opposite edges between luminium splints, nd fixing with clips. Voltile trpping ws strted fter 1 h to ensure ir homogeneity within the bgs. Voltiles were collected for 2 hours. An ETFE stopper contining two openings (0.67 mm dimeter) ws inserted into the bgs. Air contining voltiles ws sucked off from the interior ir volume of the bg through one of the openings by using miniture vcuum pump (DC12/16 NK F. Fürgut, Germny). A voltile trpping device (Prec. Chrcol filter (1.5 mg) F. Brechbuehler AG, Switzerlnd) ws 19