Limited impact of elevated levels of polyphenol oxidase on tree-feeding caterpillars: assessing individual plant defenses with transgenic poplar

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1 Oecologi (2007) 154: DOI /s z PLANT ANIMAL INTERACTIONS Limited impct of elevted levels of polyphenol oxidse on tree-feeding cterpillrs: ssessing individul plnt defenses with trnsgenic poplr Rymond V. Brbehenn Æ Christopher P. Jones Æ Lynn Yip Æ Ln Trn Æ C. Peter Constbel Received: 25 Mrch 2007 / Accepted: 13 July 2007 / Published online: 28 August 2007 Ó Springer-Verlg 2007 Abstrct Polyphenol oxidse (PPO) is commonly believed to function s n effective ntiherbivore defense in plnts. PPO is induced in plnts following herbivory, nd insect performnce is often negtively correlted with PPO levels. However, induced defenses crete numerous chnges in plnts, nd very little work hs been done to test the direct effects of PPO on insect herbivores seprtely from other chnges. This study exmined the impcts of high levels of PPO on the performnce of two species of tree-feeding cterpillrs (Lymntri dispr nd Orgyi leucostigm) on poplr. Trnsgenic PPO-overexpressing poplr (Populus tremul Populus lb) ws used s source of elevted-ppo leves, thereby controlling for the multiple effects of induction. In ddition, the impcts of treting poplr folige with high levels of purified mushroom PPO were exmined on the two cterpillr species. Contrry to expecttion, in severl cses incresed PPO levels hd no significnt effect on insect consumption or growth rtes. Although one of the mechnisms by which PPO is believed to impct herbivores is vi incresed oxidtive stress, the ingestion of lrge mounts of PPO hd Communicted by Crlos Bllré. R. V. Brbehenn (&) C. P. Jones Deprtment of Moleculr, Cellulr nd Developmentl Biology, University of Michign, Ann Arbor, MI , USA e-mil: rvb@umich.edu L. Yip L. Trn C. P. Constbel Centre for Forest Biology nd Deprtment of Biology, University of Victori, Stn CSC, P.O. Box 3020, Victori, BC, Cnd V8W 3N5 R. V. Brbehenn C. P. Jones Deprtment of Ecology nd Evolutionry Biology, University of Michign, Ann Arbor, MI , USA little or no effect on semiquinone rdicl nd oxidized protein levels in the gut contents of lymntriid cterpillrs. PPO ctivity in cterpillrs is likely limited by the low oxygen nd high scorbte levels commonly found in their gut contents. This study questions whether induced PPO functions s n effective post-ingestive defense ginst tree-feeding cterpillrs, nd indictes tht controlled, mechnistic studies re needed in other plnt herbivore systems to test for direct effect of PPO on insect performnce. Keywords Polyphenol oxidse Plnt defense Poplr Trnsgenic trees Herbivore Introduction Although polyphenol oxidse (PPO) is widely produced by plnts, the ecologicl nd physiologicl roles plyed by PPO re still not completely cler (Myer 2006). PPO hs been postulted to serve s n importnt component of plnt defenses ginst insect herbivores (Felton et l. 1989; Gtehouse 2002) nd is lso believed to function in wound heling, stress tolernce nd pthogen resistnce (e.g., Li nd Steffens 2002; Thipypong et l. 2004). When lef tissues re dmged, PPO is relesed from chloroplsts, oxidizing ortho-dihydroxy nd some trihydroxy phenolic compounds to ortho-quinones in the presence of oxygen (Rection 1). The subsequent binding of quinones to Phenolic þ 1 2 O 2! Quinone þ H 2 O ð1þ essentil mino cids in the insect gut would ruin their nutritionl vlue, thereby decresing insect performnce (Felton et l. 1989, 1992). Additionl evidence tht PPO

2 130 Oecologi (2007) 154: cts s n nti-herbivore defense hs come from studies demonstrting: (1) tht PPO ctivity is induced in mny plnts following mechnicl wounding, insect herbivory, or defense signls such s methyl jsmonte; or (2) tht insect performnce is negtively ssocited with induced PPO levels (Felton et l. 1989; Duffey nd Stout 1996; Krbn nd Bldwin 1997; Constbel nd Ryn 1998; Constbel et l. 2000). Studies testing insect performnce on plnts with incresed or decresed PPO levels hve been done primrily with species in the Solncee (tomto, potto nd tobcco) (Felton et l. 1989; Stout et l. 1998; Ren nd Lu 2006). The insect species tested hve minly been noctuid cterpillrs, but cterpillrs of Mnduc sext, Epirrit utumnt nd Mlcosom disstri, nd the beetle Leptinotrs decemlinet hve lso been exmined (Cstner et l. 1996; Redmn et l. 2001; Wng nd Constbel 2004; Ruuhol nd Yng 2006). While most of these exmples demonstrted negtive ssocitions between performnce nd PPO levels, there re lso exmples in which lck of ssocition or even positive ssocition between performnce nd PPO levels ws found (Cipollini nd Redmn 1999; Kessler nd Bldwin 2002). The interprettion of these correltive studies is gretly complicted by the fct tht concentrtions of mny secondry chemicls nd nutrients, s well s enzymes, chnge when plnt defenses re induced by herbivory (Hermsmeier et l. 2001; Mjor nd Constbel 2006). It is, therefore, impossible to evlute the effects of PPO seprtely from the effects of other potentil defenses tht re induced. To our knowledge, only one controlled study hs been published testing the direct effects of PPO on herbivore performnce (Wng nd Constbel 2004). The results of this study on Mlcosom disstri (forest tent cterpillr) were mixed, with negtive effects of elevted-ppo poplrs found only on the growth rtes of lrve rered from older egg msses. This pper reports study of the effects of ingested PPO on the performnce (reltive consumption nd growth rtes) of two species of tree-feeding cterpillrs, Lymntri dispr (gypsy moth) nd Orgyi leucostigm (whitemrked tussock moth), tht consumed poplr folige. Both re common generlist species in the deciduous forests of Europe or estern North Americ, nd both hve been used in numerous ecologicl nd physiologicl studies of plnt herbivore interctions. In this study, the mount of PPO ingested by the cterpillrs ws incresed by feeding them the folige of trnsgenic PPO-overexpressing poplr (Populus tremul Populus lb) or poplr leves treted with purified mushroom PPO, thus voiding the confounding effects of uncontrolled chnges in other defenses nd nutrients tht occur following herbivory. The increses in PPO ctivity in both types of experiments were comprble to some of the highest levels found in induced plnts (Constbel nd Ryn 1998). In ddition, we hve ssessed the effect of ingesting elevted levels of PPO on oxidtive stress in the midgut contents of the two cterpillr species. Oxidtive stress in insect herbivores cn be cused by the production of rective oxygen species, such s semiquinone nd other rective free rdicls, during the oxidtion of phenolics in the gut (Brbehenn et l. 2005) nd/or the redox cycling of quinones (Duffey nd Stout 1996). Semiquinone rdicls nd protein crbonyls were mesured s mrkers of oxidtive stress. PPO directly produces quinones, but indirectly produces semiquinone rdicls vi Rection 2 (Korytowski et l. 1987). The formtion of both of these phenolic oxidtion products would, over time, decrese levels of scorbte nd scorbyl rdicls (produced by the oxidtion of scorbic cid) vi Rections 3 5 (Ymski nd Grce 1998; Brbehenn et l. 2003). Thus, the mesurement of free rdicls with electron prmgnetic resonnce (EPR) spectrometry provides semi-quntittive mesure of the oxidtive sttus of the midgut, indicting whether PPO produces n bundnce of semiquinone rdicls (oxidtive stress), or whether the midgut contins reltively hrmless scorbyl rdicls. Protein crbonyls cn be produced by severl oxidtive mechnisms ssocited with phenolic oxidtion (e.g., Burchm nd Kuhn 1996). Quinone þ Phenolic! SQ þ SQ þ H þ Quinone þ AH! SQ þ A þ H þ SQ þ AH! Phenolic þ A A þ A þ H þ! DHA þ AH ð2þ ð3þ ð4þ ð5þ [In Rections 2 5, Phenolic nd AH represent the phenolte nd scorbte mono-nions predominntly found t physiologicl ph in the cterpillr midgut (c. ph 9 10). SQ nd A represent semiquinone nd scorbyl rdicls, respectively. DHA is dehydroscorbte, the unstble end product of scorbte oxidtion.] Mterils nd methods Plnts PPO-overexpressing P. tremul P. lb lines were produced from clone INRA717I-B4, s described previously (Constbel et l. 2000; Wng nd Constbel 2004). For

3 Oecologi (2007) 154: simplicity, lines 10, 19, nd 21 re presented s genotypes 1, 2, nd 3. High PPO ctivity in these plnts ws the result of expressing the poplr lef PtdPPO-1 gene (Constbel et l. 2000) under the control of the culiflower mosic virus double 35S promoter nd lflf mosic virus trnsltionl enhncer. Plntlets were propgted in vitro or s green cuttings, nd rooted in potting mix. Three independently trnsformed genotypes with high PPO ctivities were chosen for biossys with insects nd shipped from the University of Victori to the University of Michign. Untrnsformed prentl stock plnts served s the control genotype. Splings were grown in greenhouse in 8-l Treepots (Hummert Interntionl, Erth City, Mo.) in Sunshine potting mix (type 4), with Osmocote slow-relese fertilizer ( ) (Scotts, Mrysville, Ohio) nd wtering s needed. Growth lights with 400 W high-pressure sodium bulbs (PL Light Systems, Bemsville, Ontrio) were used from September to My (16 h light:8 h drk). Splings were grown to height of t lest 1 m over period of 3 4 months before their use in experiments. Upon completion of the experiments, ll splings were utoclved. Leves were designted by the lef plstochron index (LPI), defining the first lef with length 2 cm s LPI 0 (Wng nd Constbel 2004). Leves for experiments were cut from LPI with sterile rzor blde, using t lest three trees of ech genotype. Two to three fully expnded leves were tken per dy from ech tree, beginning with the lowest LPI. This smpling method hs no significnt effect on PPO induction in this poplr hybrid (J. Wng nd C. P. Constbel, unpublished dt). After wshing the leves for 20 min in wter bth, their surfces were blotted dry with pper towels. Lef disks were cut with cork borer (23 mm dimeter), producing pproximtely 20 disks per lef. All disks within ech genotype were mixed to rndomize potentil effects of lef position nd individul tree. Insects L. dispr nd O. leucostigm re in the fmily Lymntriide. L. dispr feeds on spring folige for single genertion, wheres O. leucostigm hs multiple genertions per yer tht feed on young or mture leves. Eggs of these species were obtined from the United Sttes Deprtment of Agriculture (Otis Air Force Bse, Mss.) nd the Cndin Forest Service (Sult Sinte Mrie, Ontrio), respectively. All lrve were rered on n rtificil diet (Brbehenn et l. 2001) in petri dishes kept in incubtors t either 18 or 23 C to regulte their developmentl rtes. Newly molted fourthinstr L. dispr nd finl-instr O. leucostigm lrve were used in most experiments. These similr-sized cterpillrs provided sufficient quntity of midgut contents for chemicl nlysis. Experiments were crried out in the winter of 2005 nd spring of 2006 on the dtes given. Chemicl nlysis Replicte lef disks cut for feeding experiments were used to mesure PPO ctivities nd the chemicl compositions of the four poplr genotypes. Smples from three to five dtes from Jnury to the end of April 2006 were selected for chemicl nlysis. This period represents the vrition cross the time when most experiments were performed. PPO ctivities were mesured in extrcts prepred from frozen ( 80 C) lef disks from control nd elevted-ppo genotypes on three dtes during the experimentl period (Constbel et l. 2000). Tnnins, low moleculr weight phenolics nd slicyltes were nlyzed primrily with high-performnce liquid chromtogrphy (HPLC) nd diode rry detector (Brbehenn et l. 2006). Ascorbic cid ws extrcted from fresh lef smples in 5% w/v metphosphoric cid (contining 1 mm EDTA) nd nlyzed with reverse-phse HPLC (Brbehenn 2003). Other nutrients were mesured in smples tht hd been lyophilized nd ground to fine powder in dentl mlgmtor (Brbehenn et l. 2004). Wter ws mesured s the difference between the fresh nd dry weights of lef disks (70 C; 2 3 dys). Lef wter content ws mesured on 14 dtes during feeding experiments (28 Jnury 21 Mrch), with five replictes per genotype on ech dte. Ascorbte concentrtions were compred in the posterior midgut contents of L. dispr (fourth instr; n = 10) nd O. leucostigm (finl instr; n = 9) on the control poplr genotype (12 June). Their posterior midgut contents were dissected, extrcted in 5% metphosphoric cid (300 ll), nd nlyzed for scorbte with reverse-phse HPLC. Mesurements of semiquinone nd scorbyl rdicls in extrcts of mid-midgut contents were mde using Bruker EMX EPR spectrometer (Bruker Instruments, Billeric, Mss.) (Brbehenn et l. 2003). Stndrd solutions of the stble free rdicl, 2,2,6,6-tetrmethyl-1-piperidinyloxy (TEMPO; Aldrich) were mde in 70% ethnol, nd two independent stndrds were run per dy (Brbehenn et l. 2006). The re of ech first derivtive spectrum ws quntified by double integrtion using WinEPR softwre (Bruker Instruments), nd double integrl vlues of smple spectr were expressed in nm TEMPO rdicl equivlents (Brbehenn et l. 2006). Severl experiments produced some spectr tht were too wek to integrte ccurtely. Therefore, for ll spectr the height of the center-field pek ws mesured (rbitrry y-xis units), nd regressions of pek height vs. double integrl vlue were used to quntify scorbyl rdicls (R 2 = 0.78) or semiquinone rdicls (R 2 = 0.96). Rdicl concentrtions in gut fluid extrcts re

4 132 Oecologi (2007) 154: presented, rther thn their concentrtions in the smple volumes, in order to compre concentrtions between extrcts nd model rection mixtures. Oxidized proteins (protein crbonyls) in cterpillr midgut contents were mesured using n enzyme-linked immunosorbnt ssy (Winterbourn nd Buss 1999). Entire midgut contents were extrcted in 400 ll of ph 6.5 phosphte buffer (200 mm, nitrogen purged), nd stored t 80 C under nitrogen tmosphere for 2 4 months before nlysis. Totl protein concentrtions in the superntnt solutions were mesured with the modified Brdford ssy (Stoscheck 1990). Aliquots of ech smple contining 7 lg of protein were plced in ech well of 96-well microtitre plte for nlysis. Protein crbonyl stndrd curves were constructed using mixtures of reduced nd oxidized bovine serum lbumin (Almdri et l. 2005). Effects of PPO on insect performnce Trnsgenic poplrs To compre the performnce of third-instr L. dispr lrve on intct plnts, newly molted lrve were weighed nd plced t rndom on seprte leves in fine nylon mesh bgs, which were tied round the petioles (10 April). Trees were mintined in the greenhouse (16-h photoperiod). Five lrve were bgged (one lrv per lef) on four trees per genotype, using LPI Trees were rotted dily to minimize positionl effects. Frss ws recovered from ech bg t the end of the third instr nd dried t 70 C. Lrve were frozen upon molting to the fourth instr, nd then dried to determine their finl weights. Lrvl fresh weights were converted to dry weights using fresh weight:dry weight rtios from six representtive lrve. Reltive consumption nd reltive growth rtes (mg/mg per dy) were clculted with men lrvl weights on dry weight bsis (Wldbuer 1968). To estimte consumption from the mss of feces produced by third-instr lrve, verge pproximte digestibilities of fourth-instr L. dispr on ech of the four poplr genotypes were used from seprte experiment (R. V. Brbehenn, unpublished dt). Fourth-instr L. dispr performnce ws compred on lef disks cut from ech of the control nd elevted-ppo poplr genotypes (28 Jnury). Newly molted lrve were rndomly ssigned to ech genotype (n = lrve/ genotype). Lrvl fresh weights were mesured nd converted to dry weights. Lrve were kept in individul 35-ml plstic cups in 23 C incubtor (16 h light:8 h drk). Leves nd lef disks were cut nd prepred s described bove. Fresh weights of lef disks fed to ech lrv were recorded, nd fresh weight:dry weight rtios of representtive lef disks (n = 5) from ech genotype were used to convert food weights to dry weights to mesure consumption. The difference between the dry weight of the food nd the dry weight of the uneten remins ws defined s the mount consumed. Fresh lef disks were provided dily nd retined their turgidity on moist filter ppers. Fecl pellets were collected dily, pooled for ech individul lrv nd dried (70 C). Upon molting to the fifth instr, lrve were frozen, dried nd weighed. Consumption nd growth rtes were mesured on dry weight bsis, s described bove. An identicl experiment ws performed on finl-instr O. leucostigm (14 Mrch). PPO-coted leves To exmine the effect of PPO dded to poplr leves (control genotype) on lrvl performnce, fourth-instr L. dispr were fed lef disks (5 Februry) coted with 20 ll of 50% cetone suspension of mushroom PPO. Control lef disks were treted with 50% cetone. The solvent contined 5.7 mg sucrose/ml, s in other lef-coting experiments. The PPO suspension or control solvent ws spred evenly on lef disks using n djustble pipetter, with the pipette tip held horizontlly to void dmging the lef surfce. PPO ws pplied to lef disks t 4,467 tyrosinse U/g lef fresh weight, ccording to the tyrosinse ctivities reported by the mnufcturers. Sigm PPO (1,530 tyrosinse U/mg enzyme preprtion) ws used for three dys, followed by Worthington PPO (836 tyrosinse U/mg enzyme preprtion) on the finl dy. When PPO ctivity ws lter mesured in the commercil PPO preprtions using the sme methods tht we used to mesure PPO in leves (see Chemicl nlysis bove), these ctivities were found to differ from the expected levels. Thus, PPO ctivities were incresed fourfold (by 59 PPO U/g fresh weight) for 3 dys, nd pproximtely eightfold (by 103 PPO U/g fresh weight) on the finl dy. Lrve were weighed, ssigned t rndom to the control (n = 10) or PPO (n = 13) tretment groups, nd fed weighed mount of poplr lef disks. Consumption nd growth rtes were mesured on dry weight bsis, s described bove. Preliminry tests showed tht 90% of the dded PPO ctivity could be recovered from treted lef disks 30 min fter the cotings were dried (R. V. Brbehenn, unpublished dt). Becuse poplr PPO is stble during its pssge through the guts of some cterpillr species (Wng nd Constbel 2004), we ssumed tht the dded PPO protein hd negligible nutritionl effect. Phenolic-coted leves If PPO ctivity in poplr-feeding cterpillrs ws limited by low substrte concentrtion, then incresing substrte

5 Oecologi (2007) 154: levels would potentilly increse the impct of PPO on cterpillr performnce. To test this possibility, finlinstr O. leucostigm were plced t rndom in one of four tretments: control genotype leves (solvent-treted), elevted-ppo genotype leves (solvent-treted), control genotype leves coted with chlorogenic cid (4% dry weight), or elevted-ppo leves coted with chlorogenic cid (4%). Chlorogenic cid ws chosen s PPO substrte becuse it is one of the most bundnt phenolics in poplrs (see Results ), nd is one of the commonly recognized substrtes for PPOs (e.g., Felton et l. 1989). Chlorogenic cid (Sigm) ws dissolved (30.4 mg/ml) in 50% queous cetone contining 5.7 mg sucrose/ml to promote feeding. A 20-ll liquot of this solution ws coted on lef disks (cut nd mixed s described bove). Lef disks from ll three elevted-ppo genotypes were mixed in equl proportion. After the solvent dried on the disk surfces (c. 5 min), disks were fed to ech lrv (n = 9 13/tretment). The number of lef disks fed to lrve ws incresed from three to six s consumption incresed from dy to dy. Consumption nd growth rtes were mesured grvimetriclly (beginning Mrch 15). Effects of PPO on oxidtive stress Trnsgenic poplrs Seprte experiments were performed to mesure either free rdicls or oxidized proteins in midgut contents. Cterpillrs were plced t rndom on lef disks from one of the four poplr genotypes (n = 12 16/genotype). Lrve were kept seprtely in 35-ml cups in 23 C incubtor. On the third dy, lrve were llowed to feed on fresh disks for t lest 1 h, following which they were chilled ( 20 C, 6 min) nd dissected under dissecting microscope. To mesure free rdicls, midgut contents (20 30 mg) were plced in 300 ll of ph 10 crbonte buffer (nitrogen purged) under nitrogen tmosphere, weighed to the nerest 0.1 mg, shken vigorously, nd centrifuged (c. 8,000 g, 1 min, mbient temperture). A 200-ll liquot of the superntnt solution ws used for EPR spectrometry, s described bove. All smples were scnned within 4 5 min from the time the gut contents were dissected, permitting ccurte comprisons of scorbyl or semiquinone rdicl concentrtions in gut fluid smples (Brbehenn et l. 2003, 2005). Free rdicls were mesured in L. dispr lrve on 17 November (2005), nd in O. leucostigm on 14 Februry. Oxidized proteins (protein crbonyls) were mesured in the midgut contents of O. leucostigm on 26 April nd in L. dispr 7 December (2005) (n = 12 16/tretment), s described bove. PPO-coted leves Purified mushroom PPO ws pplied to lef disks t 4,467 tyrosinse U/g lef fresh weight, s described bove. For L. dispr, lef disks were cut from three control trees, nd mixed to rndomize potentil lef position or tree effects (21 April). PPO (Worthington, 836 tyrosinse U/mg enzyme preprtion) ws used to increse folir PPO ctivity by 103 PPO U/g fresh weight, or pproximtely sevenfold increse. This experiment ws repeted (18 My) with Sigm PPO (3,900 tyrosinse U/mg enzyme preprtion), resulting in n increse of 139 PPO U/g fresh weight. A similr experiment ws performed on O. leucostigm (11 July) by coting lef disks with PPO (Sigm, 3,320 tyrosinse U/mg enzyme preprtion). Ingested PPO ctivities were incresed by 140 PPO U/g fresh weight (pproximtely tenfold). Free rdicl levels were mesured in cterpillr midgut contents with EPR spectrometry s described bove (n = 8 12/tretment). Phenolic-coted leves L. dispr lrve were rndomly ssigned to feed on lef disks from ech genotype tht were coted with chlorogenic cid (4% dry weight) (2 December). This experiment ws conducted s described bove for exmining the performnce of O. leucostigm, with the exception tht ech of the four genotypes ws tested. Semiquinone rdicl levels were mesured in cterpillr midgut contents with EPR spectrometry. No concurrent mesurements of rdicl levels in lrve tht ingested untreted lef disks were mde. Bsed on rdicl levels observed in ten other EPR experiments on L. dispr on the sme hybrid poplr (R. V. Brbehenn, unpublished dt), it ws ssumed tht either very low levels of semiquinone rdicls or scorbyl rdicls would be present. For comprison with the effects of chlorogenic cid tretment, the results of the nerest comprble experiment re presented (17 November; n = 10 17/genotype). Effect of scorbte concentrtion on PPO ctivity The potentil of scorbte to limit PPO ctivity ws compred in conditions found in cterpillr midgut fluid extrcts (pproximtely 7% v/v dilution) nd in conditions tht more closely pproximte in vivo (undiluted) scorbte levels. Rection mixtures for EPR were prepred with 290 ll of ph 10 crbonte buffer (low oxygen), to which 10 ll of low-oxygen ph 4 double-distilled wter (for low scorbte tretment) or 10 ll of 4.5 mm scorbte in ph 4 double-distilled wter ws dded. It ws clculted

6 134 Oecologi (2007) 154: tht the scorbte levels in rection mixtures rnged from 62 to 124 lm (with no scorbte dded) nd from 212 to 274 lm (150 lm scorbte dded), bsed on scorbte mesurements from the midguts of L. dispr (see below). The mid-midgut contents (c. 25 mg) of fourth- nd fifthinstr L. dispr lrve recently feeding on poplr (control genotype) were extrcted in either the scorbte-free or scorbte-contining ph 10 buffer (nitrogen-purged), mintined under nitrogen tmosphere. A 200-ll liquot of the extrct ws spiked with 10 ll of chlorogenic cid solution (14 mm in 70% low-oxygen ethnol) or doubledistilled, low-oxygen wter ( CGA tretment), nd with 10 ll of PPO solution (0.2 mg PPO/ml low-oxygen double-distilled wter) or double-distilled, low-oxygen wter ( PPO tretment). PPO (Sigm) hd n ctivity of 1,530 tyrosinse U/mg protein. Rection mixtures were mixed rpidly with 200-ll pipetter, nd trnsferred to flt cell in the EPR cvity. EPR spectr were recorded beginning pproximtely 30 s from the strt of the rection for 3 min (two scns). All prmeters were identicl to those described bove for other EPR experiments. Four combintions of chlorogenic cid nd PPO tretments were tested, with totl of 3 7 replictes/tretment exmined over 2-dy period. These experiments were designed to qulittively test the hypothesis tht scorbte levels in the midgut contents of cterpillrs cn inhibit PPO ctivity. Sttisticl nlysis Lef chemicl composition ws compred cross tree genotypes nd smpling dtes with two-wy ANOVA (PROC MIXED) (SAS 2003). One smple representing ech genotype-by-dte combintion ws nlyzed, nd results were pooled cross dtes to simplify their presenttion. Seprte experiments were performed to mesure the performnce for ech cterpillr species, nd to mesure levels of free rdicls nd oxidized proteins in their midgut contents. Individul lrve were used s replictes in ll experiments. Reltive growth nd consumption rtes were compred cross tree genotypes with nlysis of covrince (ANCOVA) (PROC MIXED). Models for nlyzing performnce used tree genotype s the min effect. In comprisons of L. dispr performnce on intct leves, the effects of lef position nd lef genotype interctions were lso tested. ANCOVA models used to compre insect performnce cross poplr genotypes were s follows: reltive growth rtes (mg/mg per dy) were compred using growth rte s the dependent vrible nd initil dry weight s the covrite. Reltive consumption rtes (mg/mg per dy) were compred using consumption rte s the dependent vrible nd initil dry weight s the covrite. In ll cses, models testing for significnt interction between the dependent vrible nd covrite were first tested to confirm tht the regression slopes were prllel. Pirwise differences between lrve on ech tree genotype were exmined by differences of lest squres mens (P = 0.05 for priori comprisons or djustment with the Tukey Krmer method for unplnned comprisons) (SAS 2003). To test the hypothesis tht lrve on the control genotype differed from lrve on the three PPO genotypes, these two groups were compred with contrsts (weighted s 3, 1, 1, 1) (PROC MIXED). Levels of free rdicls nd oxidized proteins were compred cross tree genotypes for L. dispr nd O. leucostigm with one-wy ANOVA nd contrsts, s described bove. The normlity of residuls ws tested with PROC UNIVARIATE (SAS 2003). Where necessry, log or squre root trnsformtions were used to normlize residuls. Dt which could not be trnsformed to meet the ssumptions of ANOVA (rdicl levels in the midgut contents L. dispr on PPO- nd chlorogenic cid-treted lef disks) were nlyzed with Kruskl Wllis tests (Wilkinson 2000). Results PPO ctivities were substntilly greter in ll the elevted- PPO poplr genotypes, lthough the mgnitude of the increse vried by experimentl dte. PPO ctivities in genotypes PPO 1, PPO 2, nd PPO 3 were elevted 28-, 29- nd 27-fold, respectively, on 28 Jnury, five-, five- nd ninefold on 16 Mrch, nd 12-, 14- nd 26-fold on 25 April. Control genotype PPO levels were 10, 6 nd 18 U/g fresh weight, respectively, on ech of the exmined dtes. Even t their lowest levels of increse (i.e., increses of 70 U/g fresh weight), elevted-ppo poplr folige contined higher PPO ctivities thn would be encountered by herbivores feeding on wide vriety of other plnt species (Constbel nd Ryn 1998). By contrst, the elevted-ppo genotypes showed no significnt differences in vriety of chemicl trits tht could ffect the performnce nd gut biochemistry of cterpillrs: phenolics, slicyltes, scorbte, protein, nonstructurl crbohydrtes nd wter (Tble 1). Three min groups of phenolics were identified nd quntified: chlorogenic cid nd its derivtives (three compounds totling 0.7% dry weight), flvonoid glycosides (nine compounds totling 0.4% dry weight) nd coumroylquinic cid nd its derivtives (three compounds totling 0.8% dry weight). No condensed or hydrolyzble tnnins were detected. Slicyltes (or phenolic glycosides ) show little utoxidtion t ph 10 compred with phenolic compounds (R. V. Brbehenn, unpublished dt), re not substrtes for PPO, nd re thus reported seprtely from phenolic compounds. Nine slicyltes were detected, with slicortin nd tremulcin comprising pproximtely

7 Oecologi (2007) 154: Tble 1 Chemicl composition of control genotype nd elevted-polyphenol oxidse (PPO) poplr genotypes. DW Dry weight, FW fresh weight Genotype Phenolics b (% DW) Slicyltes c (% DW) Ascorbte (lmol/g FW) Protein (% DW) Crbohydrtes d (% DW) Wter (% FW) Control 1.2 ± ± ± ± ± ± 0.3 PPO ± ± ± ± ± ± 0.5 PPO ± ± ± ± ± ± 0.4 PPO ± ± ± ± ± ± 0.5 Significnce of effects Genotype P = P = P = P = P = P = Dte P = P = P = P = P = P \ One smple per genotype ws nlyzed from three dtes between Jnury nd April 2006 for phenolics nd slicyltes, four dtes for scorbte, five dtes for protein, four dtes for crbohydrtes nd 14 dtes for wter b All mesurble low moleculr weight phenolics were summed: chlorogenic cid nd its derivtives, coumrylquininic cid nd its derivtives, nd flvonoid glycosides. No tnnins were detected c Slicyltes included primrily tremulcin nd slicortin, but lso seven unidentified slicyltes present t % DW ech d Crbohydrtes were present in rtio of pproximtely 4:1:8:1 for glucose, fructose, sucrose nd strch, respectively 70% of these compounds. Severl nutrients vried significntly through time in ech of the genotypes, presumbly s result of tree ge or environmentl chnges. Ascorbte rnged from low level of pproximtely 7 lmol/g fresh weight in Jnury nd Februry, to high of 10 lmol/g in lte April. Similrly, totl nonstructurl crbohydrtes vried from n overll verge of pproximtely 6% dry weight in Jnury nd Februry, to high of pproximtely 9% dry weight in Mrch nd lte April. The control genotype nd elevted-ppo poplrs displyed no obvious differences in growth rtes or morphology, s shown previously (Wng nd Constbel 2004). To test the effects of PPO on cterpillrs feeding on splings, third-instr L. dispr lrve were enclosed seprtely in mesh bgs on intct leves. After 4 5 dys of feeding, neither consumption nor growth rtes differed significntly between lrve on the control genotype nd the three elevted-ppo genotypes (contrst P = nd P = 0.791, respectively) (Fig. 1, b). Both consumption nd growth rtes were higher on the youngest lef thn on the next older four leves (P \ nd P \ 0.050, respectively), but no significnt lef genotype interctions were found for consumption (P = 0.855) or growth rtes (P = 0.957). Becuse PPO ctivities re not significntly induced by mechnicl dmge in the poplr hybrid used in this work (C. P. Constbel, unpublished dt), PPO levels in the control nd elevted-ppo genotypes were ssumed to remin unchnged during this experiment. In fourth-instr L. dispr tht fed on lef disks, reltive growth rtes were decresed by 10 16% on elevted-ppo poplr leves compred with control genotype leves (contrst P \ 0.001) (Fig. 1b). Consumption rtes were not significntly different between lrve on the four poplr genotypes (P = cross genotypes; contrst P = 0.064) (Fig. 1). When high levels of PPO were coted A RCR (mg/mg/dy) B RGR (mg/mg/dy) rd Instr 3rd Instr Control PPO 1 PPO 2 PPO 3 Control PPO 1 PPO 2 PPO 3 on control genotype leves, no effects on the consumption or growth rtes of fourth instr L. dispr were observed (P = nd P = 0.810, respectively) (Fig. 2). The performnce of O. leucostigm lrve ws unffected by feeding on the elevted-ppo poplr genotypes b 4th Instr b 4th Instr Fig. 1 Reltive consumption rtes (RCR) nd b reltive growth rtes (RGR) of third-instr Lymntri dispr on control nd elevtedpolyphenol oxidse (PPO) poplr splings, nd fourth-instr L. dispr on control nd elevted-ppo poplr lef disks. Different letters bove brs designte P \ 0.05 b

8 136 Oecologi (2007) 154: Rte (mg/mg/dy) Control PPO-coted b b A Rte (mg/mg/dy) Control PPO 1 PPO 2 PPO RCR RGR Fig. 2 RCR nd RGR of fourth-instr L. dispr on poplr lef disks (control genotype) coted with solvent or PPO. For bbrevitions, see Fig. 1 (Fig. 3). Consumption rtes did not differ significntly cross genotypes (P = 0.747) or in the contrst between control nd elevted-ppo genotypes (P = 0.369). Growth rtes were lso similr cross genotypes (P = 0.369) nd when control nd elevted-ppo genotypes were contrsted (P = 0.400). If PPO from elevted-ppo poplrs hd little effect on cterpillrs becuse PPO is substrte limited in the gut, then coting leves with chlorogenic cid (4% dry weight) should produce significnt PPO effects on cterpillr performnce. When the performnce of O. leucostigm ws compred on the control genotype nd elevted-ppo leves, ech with nd without dded chlorogenic cid, no significnt genotype chlorogenic cid interction ws found for consumption or growth rtes (P = nd P = 0.454, respectively) (Fig. 3b). The lck of significnce of the interction effect is of primry interest becuse it indictes tht dded chlorogenic cid hd no more effect on elevted-ppo leves thn it did on control genotype leves. Growth rtes of O. leucostigm on elevted-ppo leves showed nerly significnt 6.5% decrese compred with lrve on the control genotype leves (P = 0.052), while consumption rtes did not differ (P = 0.504). Chlorogenic cid itself hd no pprent effect on consumption or growth rtes (P = nd 0.290, respectively). The potentil for PPO to produce oxidtive stress within the insect gut ws exmined by mesuring free rdicls nd oxidized proteins. L. dispr tht fed on control nd elevted-ppo poplrs contined similr, low levels of semiquinone rdicls in their midgut contents (58 ± 11 nm in control genotype; 62 ± 7 nm cross PPO genotypes; P = 0.594). There ws lso no significnt effect of elevted-ppo poplr folige on protein crbonyl levels in L. dispr, which verged 1.0 ± 0.04 nd 1.0 ± 0.03 nmol/ mg protein in lrve on the control genotype nd cross the PPO genotypes, respectively (P = 0.198). B Rte (mg/mg/dy) RCR RCR Control-CGA PPO-CGA Control+CGA PPO+CGA RGR b b RGR Fig. 3 RCR nd RGR of finl-instr Orgyi leucostigm on control genotype nd elevted-ppo poplr lef disks. b RCR nd RGR of finl-instr O. leucostigm on control genotype nd elevted-ppo genotype poplr lef disks (pooled PPO genotypes) with (+) or without ( ) dded chlorogenic cid (CGA; 4% dry weight). For other bbrevitions, see Fig. 1 In the midgut contents of O. leucostigm, reltively high scorbyl rdicl concentrtions were detected in lrve on ll genotypes. However, lrve tht consumed control genotype PPO poplr leves hd significntly higher level of scorbyl rdicls (81 ± 6 nm) thn did lrve on the elevted-ppo genotypes (56 ± 4 nm cross genotypes) (contrst P = 0.002). As would be expected from the presence of high scorbyl rdicl (nd scorbte) levels in the midgut contents, there ws no significnt difference between protein crbonyl levels in O. leucostigm on the control genotype (1.4 ± 0.1 nmol/mg protein) or elevted- PPO genotypes (1.5 ± 0.1 nmol/mg protein) (P = 0.888). When poplr leves were treted to increse PPO levels sevenfold (using Worthington PPO in April), scorbyl rdicls in L. dispr midgut extrcts decresed 40% from 177 ± 11 to 107 ± 15 nm (P = 0.003). However, this result ws not repetble in n experiment 1 month lter using Sigm PPO with higher specific ctivity (22 ± 5 vs. 14 ± 3 nm; P = 0.324). In O. leucostigm, even tenfold increse in PPO (Sigm) produced no significnt effects on scorbyl rdicl levels, verging 161 ± 16 nd 151 ± 3 nm in extrcts from lrve on control nd elevted-ppo genotypes, respectively (P = 0.465). In no cse

9 Oecologi (2007) 154: ws there shift towrds the production of semiquinone rdicls (oxidtive stress) from PPO ctivity. If ingested PPO ws limited by low levels of phenolic substrtes in poplr folige (Tble 1), then the ingestion of high levels of chlorogenic cid should hve produced significnt PPO effect on semiquinone rdicl levels (vi Rections 1 nd 2). However, the midgut contents of L. dispr lrve tht fed on elevted-ppo leves coted with chlorogenic cid contined similr levels of semiquinone rdicls compred with lrve tht fed on control genotype leves tht were coted with chlorogenic cid (Fig. 4) (contrst P = 0.681). In the bsence of dded chlorogenic cid, t most, only low levels of semiquinone rdicls were produced in L. dispr on control nd elevted-ppo genotypes. Midgut fluid extrcts from L. dispr tht were spiked with chlorogenic cid (0.64 mm finl concentrtion) produced oxidtive stress, i.e., switched from scorbyl rdicls to semiquinone rdicls (Fig. 4b). Contrry to expecttion, the ddition of PPO to extrcts lso cused switch from A Semiquinone rdicls (nm) B R dicl concentrtion (nm ) b b C-CGA PPO-CGA C+CGA PPO+CGA Poplr genotypes +/- 4% CGA ~90 µm scorbte ~240 µm scorbte SQ* SQ* SQ* AA* AA* AA* AA* AA* -PPO/-CGA +PPO/-CGA -PPO/+CGA +PPO/+CGA Spiked gut fluid tretments Fig. 4 Composite view of semiquinone rdicl concentrtions in the midgut contents of fourth-instr L. dispr fter feeding on control or elevted-ppo genotypes without dded CGA (4% dry weight) (November 17) or on control or elevted-ppo genotypes treted with CGA (4% dry weight) (December 2). b Effect of scorbte concentrtion on PPO ctivity in rection mixtures contining L. dispr midgut fluid from poplr-feeding lrve. ph 10 buffered extrcts were spiked with PPO nd/or CGA. AA* Ascorbyl rdicls, SQ* semiquinone rdicls; for other bbrevitions, see Figs. 1 nd 3 scorbyl to semiquinone rdicls, the mounts of which were gretly incresed by dding both PPO nd chlorogenic cid together. However, when scorbte ws present t n verge concentrtion of pproximtely 240 lm it inhibited both the oxidtion of dded chlorogenic cid nd the bility of PPO to generte high stedy-stte levels of semiquinone rdicls. Ascorbte concentrtions in the posterior midgut contents of L. dispr (585 ± 51 lm) nd O. leucostigm (934 ± 81 lm) were substntilly greter thn 240 lm, nd levels in O. leucostigm were significntly greter thn those in L. dispr (P = 0.002). Discussion Numerous correltive studies hve been done on the potentil importnce of PPO s n ntiherbivore defense in both ecologicl nd griculturl systems. Yet few studies hve exmined the efficcy of the induced levels of this puttive defense tht commonly follow herbivory. While PPO is undoubtedly n importnt component of the suite of defense-relted proteins tht re induced in poplr nd mny other plnts by insect herbivores, the results of this study suggest tht the efficcy of PPO s defense ginst cterpillrs my be much weker thn hs been believed. Overll, incresed levels of either poplr PPO or mushroom PPO produced only limited effects on the performnce nd oxidtive stress in two tree-feeding cterpillr species. In one striking exmple, L. dispr lrve fed continuously on the intct leves of elevted-ppo splings with no effect on their consumption or growth rtes. If PPO remined ctive in cterpillrs, it would be expected to produce substntil increse in semiquinone rdicls in their midgut contents, s ws observed in ph 10 buffer (e.g., Fig. 4b; low scorbte series). Insted, we typiclly observed scorbyl rdicls (produced by the oxidtion of scorbic cid) in cterpillr midguts. In no cse did the ingestion of incresed levels of PPO overwhelm the scorbte ntioxidnt defense system, producing incresed levels of semiquinone rdicls (oxidtive stress). These results further support the importnce of the scorbte ntioxidnt defense system in the gut contents of cterpillrs (Felton nd Duffey 1992; Brbehenn et l. 2001, 2003, 2005). The lck of effect of induced levels of PPO on the formtion of protein crbonyls lso suggests tht there ws not physiologiclly significnt increse in phenolic oxidtion by ingested PPO in the gut contents of L. dispr nd O. leucostigm cterpillrs. Nevertheless, the results of this study were mixed, nd some results could indicte occsionl negtive effects of PPO on cterpillrs. In one experiment, the growth rtes fourth-instr L. dispr lrve were 11 16% lower on the elevted-ppo genotypes compred with the control

10 138 Oecologi (2007) 154: genotype, nd in one experiment the growth rtes of O. leucostigm were 6.5% lower on elevted-ppo poplr genotypes thn on the control genotype. The lower levels of scorbyl rdicls in the midgut contents of O. leucostigm tht fed on elevted-ppo poplr leves could lso indicte incresed PPO ctivity, either in the lef tissues during ingestion or in the gut contents following ingestion. However, where there ws decrese in performnce in lrve on the elevted-ppo genotypes, the lck of chnge in biochemicl mrkers of PPO ctivity mkes it uncler wht mechnism(s) could ccount for n effect of PPO on performnce. For exmple, lower growth rtes of fourthinstr L. dispr on elevted-ppo genotypes were not consistent with the pprent lck of PPO ctivity in their gut contents. If PPO is n effective post-ingestive plnt defense, one would expect consistent congruence between PPO levels, oxidtive stress nd insect performnce. In ddition, one might lso expect greter impct on performnce by high levels of PPO. In previous studies using different insect species, decreses in growth rtes ttributed prtilly or entirely to PPO hve commonly been on the order of 20 50% (Felton et l. 1989; Cstner et l. 1996; Cipollini nd Redmn 1999; Wng nd Constbel 2004; Ren nd Lu 2006). At lest four fctors potentilly limit PPO ctivity in the gut contents of cterpillrs: low levels of oxygen, high levels of scorbte, high ph, nd limited lef tissue mcertion. Ambient oxygen levels of bout 150 mm Hg fll quickly to between 0 nd 6 mm Hg in the foreguts nd midguts of most cterpillrs (Johnson nd Brbehenn 2000). It remins to be shown whether higher oxygen levels in the unusully lrge foreguts of L. dispr (e.g., 32 mm Hg in lrve fed rtificil diet) permit higher levels of oxidtion in this region. Ascorbte in in vitro rection mixtures t pproximtely lm, cn chemiclly reduce quinones nd semiquinone rdicls, thereby limiting the effectiveness of PPO s n oxidtive defense (Jnovitz- Klpp et l. 1990; Felton nd Duffey 1992). Ascorbte levels mesured in the midgut contents of both cterpillr species in this study were well bove these lower limits. The high ph found in cterpillr midguts could lso decrese the ctivities of ingested PPO by over 50% (Felton et l. 1989; Wng nd Constbel 2003). Finlly, mny cterpillrs feed by rpidly snipping nd swllowing lef pieces without mcerting them (Brbehenn 1992). Thus, ingested lef tissues not only pss into low-oxygen environment in mtter of seconds, but much of the ingested lef tissues remin structurlly intct, seprting PPO nd phenolic substrtes until cell membrnes rupture inside the gut. In ddition, the induction of high levels of PPO might not produce incresed PPO ctivity in cterpillrs if folige contined indequte mounts of phenolic substrtes. The ddition of chlorogenic cid to poplr leves provided lrge mount of substrte for PPO in the gut contents of L. dispr lrve. However, the high levels of PPO in trnsgenic trees did not increse the oxidtion of the dded phenolics, nd insted produced similr semiquinone rdicl levels in lrve on the control nd elevted-ppo genotypes. This result suggests tht induced levels of PPO in poplr leves would not increse the level of defense ginst poplr-feeding cterpillrs, t lest not s result of post-ingestive effects on oxidtive stress. It remins possible tht other mrkers of PPO ctivity, such s levels of quinones or quinone-bound proteins, would provide more conclusive evidence for PPO ctivity in the guts of cterpillrs thn mesures of oxidtive stress, but in either cse substntil impct on herbivore fitness would be expected if PPO is n effective plnt defense. In the cse of O. leucostigm, there ws no indiction of detrimentl synergistic effect on performnce from incresing both PPO nd substrte levels (Fig. 3b). This finding, gin, strongly suggests tht ingested PPO ws not limited by lck of phenolic substrtes in poplr leves. Both the reltively low levels of semiquinone rdicls formed nd the similrity of these levels cross low nd high PPO genotypes (Fig. 4) suggest tht the chlorogenic cid-treted leves produced incresed non-enzymtic phenolic oxidtion (e.g., with Fe 3+ or O 2 ). Phenolic oxidtion is promoted by the high ph of cterpillr midguts (c. ph 9 10) when either low levels of scorbte, high levels of phenolics, or highly rective types of phenolics re present (Brbehenn et l. 2003, 2005, 2006). If PPO-ctlyzed rections in the gut contents of cterpillrs re limited by low oxygen levels nd high scorbte levels, previous work on mechnisms of ctivity of PPO in cterpillrs would need to be re-ssessed. In the seminl studies on PPO s n ntiherbivore defense, mbient oxygen levels nd scorbte-free buffers were used to model the effects of PPO in cterpillr gut contents (e.g., Felton et l. 1989, 1992). In ddition, previous estimtions of the extent of PPO-ctlyzed rections in the gut were bsed on levels of protein-bound quinones in the feces (Felton et l. 1989). Since PPO survives trnsit through the guts of some cterpillrs nd remins ctive in their feces (Wng nd Constbel 2004), it is likely tht mesurements of quinonebound proteins in fecl smples were incresed both by enzymtic nd non-enzymtic oxidtion t mbient oxygen levels. The need to confirm previous work on oxidtion products in fecl pellets is lso supported by our observtions tht the bright green contents of the midgut rpidly turn brown when exposed to ir (i.e., oxygen) either in the rectum or fter fecl pellets re produced (R. V. Brbehenn, personl observtion). Indirect support for PPO s n ntiherbivore defense hs come from the observtion tht PPO is co-induced with suite of nti-nutritive proteins, ll of which re upregulted

11 Oecologi (2007) 154: by herbivore-stimulted signling pthwy (e.g., Bergey et l. 1996; Ren nd Lu 2006). In mny poplrs, lef dmge induces PPO long with other suspected nd confirmed defense proteins, such s protese inhibitors (Mjor nd Constbel 2006). However, in mny species there is some overlp in signling of insect nd pthogen defense responses (e.g., Mewis et l. 2006). Thus, it is possible tht PPO contributes to defense ginst plnt pthogens. The importnce of PPO s n ntimicrobil defense is supported by the observtion tht resistnce to pthogenic bcteri is directly ffected by PPO levels in tomto (Li nd Steffens 2002; Thipypong et l. 2004b), nd tht PPO is induced by fungl pthogens (e.g., Rj et l. 2006). Therefore, it is possible tht one role of PPO is to inhibit opportunistic pthogens tht enter leves t wounds creted by herbivores. We re currently investigting pthogen resistnce in elevted-ppo poplr. While the conclusions of our work on lymntriid cterpillrs do not necessrily pply to ll other insect herbivores, this study does suggest tht controlled, mechnistic studies re needed to test for direct effect of PPO on insect performnce in these other systems. Plnt herbivore interctions in which PPO my be more likely to function s n ntiherbivore defense include those in which the herbivore species re more susceptible to plnt oxidtive defenses thn re lymntriid cterpillrs (e.g., M. disstri) (Wng nd Constbel 2004; Brbehenn et l. 2005). It is lso possible tht first-instr lrve re more susceptible to the potentil effects of PPO, lthough the seminl work of Felton nd collegues (1989) suggested tht both neonte nd mture lrve would be susceptible. There my lso be vrition in the effectiveness of PPO cross plnt tx depending on its site of storge. For exmple, the rpid relese of lrge mounts of PPO from the glndulr trichomes of mny species in the Solncee (Yu et l. 1992) could produce greter PPO ctivity during ingestion thn does the slower relese of smller mounts of PPO from mesophyll nd other lef tissues. Acknowledgements This project ws supported by the Ntionl Reserch Inititive of the USDA Coopertive Stte Reserch, Eduction nd Extension Service, grnt number to R. V. B. nd C. P. C. We thnk Juh-Pekk Slminen for phenolics nlysis, KimHng Dinh for protein crbonyl nlysis, Rsik Rngnthn for scorbte nlysis, Brd Binges for help with plnt cre, Ken Guire for sttisticl consulttion, nd Michel M. Mrtin nd Gry Felton for excellent comments on the mnuscript. All experiments were performed in complince with current lws of the United Sttes. 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