Do Different Casein Concentrations Increase the Adverse Effect of Rutin on the Biology of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)?

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1 774 Septemer - Octoer 21 PEST MANAGEMENT Do Different Csein Concentrtions Increse the Adverse Effect of Rutin on the Biology of Anticrsi gemmtlis Hüner (Lepidopter: Noctuide)? M r i n C S lv d o r 1,2, Ar l i n d o L Bo i ç Jr 1, M r i C N d e Ol i v e i r 2, Jo s é P d Gr ç 1,2, Dé o r M d Si l v 2,3, Cl r B Ho f f m n n-c m p o 2 1 Univ Estdul Pulist, Vi de Acesso Prof. Pulo Donto Cstellne s/nº, , Joticl, SP, Brsil; mrincloss@yhoo.com.r, oicjr@fcv.unesp.r 2 Emrp Soj, CP 231, , Londrin, PR, Brsil; hoffmnn@cnpso.emrp.r 3 Univ Federl do Prná, CP 192, , Curiti, PR, Brsil; deor@cnpso.emrp.r Edited y José Roerto Slvdori EMBRAPA Neotropicl Entomology 39(5): (21) ABSTRACT - The flvonoid rutin is recognized s plying n importnt role in the protection of plnts ginst lepidopterns. Biossys with this compound re generlly crried out using rtificil diets. Proteins of high energy vlue, such s csein, re importnt ingredients of insect rtificil diets s source of essentil mino cids. However, such proteins cn generlly increse the llelochemicl ctivity. Our ojective ws to evlute the effects of rutin on lrve of the velveten cterpillr Anticrsi gemmtlis Hüner y incorporting this llelochemicl into diets with different concentrtions of csein. Three csein concentrtions (, 7 g, or 14 g) comined with none,.65%, or 1.3% of rutin were dded to the rering diet nd offered to the lrve from htching to puption. Rutin negtively ffected lrvl development, the mount of food consumed, nd pupl weight of A. gemmtlis. These negtive effects were clerly seen in insects fed on diets with 7 g of csein to which ny concentrtion of rutin ws dded. The effects of rutin when dded to the diets without csein were stronger thn in diets contining suitle mount of csein (14 g). The greter negtive effects of rutin in diets contining suoptiml concentrtions of csein indicte tht csein cn increse the effects of rutin only when the diets re nutritionlly unsuitle for insect development. KEY WORDS: Flvonoid, velveten cterpillr, iologicl prmeter Flvonoids re chemicl compounds widely distriuted in plnts (Simmonds 21). They ply n importnt role in plnt defense ginst microorgnisms nd insects (Dixon & Steele 1999) nd cn e ttrctive, deterrent, or toxic to pests (Hrorne & Gryer 1993). The flvonoid rutin (quercitin 3-O-rhmnosylglucoside) ws detected in the soyen genotype PI (Hoffmnn-Cmpo et l 21, Piuelli et l 25), which hs een widely used in reeding progrms s source of resistnce ginst defoliting insects. Rutin hs shown ntiiotic nd/or ntinutritionl effects on mny chewing insects (Stmp & Skrool 1993), including the velveten cterpillr, Anticrsi gemmtlis Hüner (Hoffmnn-Cmpo et l 26), mjor defoliting pest of soyens in Brzil. For insect optiml growth in rtificil diets, proteins or mino cids re required in high concentrtions (Pnizzi & Prr 1991). Among proteins, csein hs een widely used in rtificil rering diets ecuse it contins ll the essentil mino cids, is solule in wter, nd does not cogulte or precipitte fter heting (Prr 1979). Csein lso contins trces of importnt sustnces such s ftty cids, cholesterol, sugrs, vitmins, nd minerls (Vnderznt 1966). The effect of plnt-defense chemicl compounds on the iology nd ehvior of insects is rrely studied independently. According to Duffey & Stout (1996), the ingredients used in rtificil diets for insect rering cn increse the impct of chemicl compounds relted to plnt defense. They lso rgued tht high-energy proteins such s csein, which is one of the ingredients of the rtificil diet of the velveten cterpillr, when ssocited with chlorogenic cid, increses its negtive influence on lrvl growth in Lepidopter. Chlorogenic cid nd rutin re phenolic compounds, nd informtion on the possiility of interction etween csein nd rutin is essentil to confirm the reliility of results otined on the effects of flvonoids on insects when tested on rtificil diets. Therefore, our min ojective ws to evlute if csein cn increse the effects of rutin on the iologicl development of A. gemmtlis. Mteril nd Methods Anticrsi gemmtlis ws mintined on n rtificil diet sed on ens, soyen protein, whet germ, csein, nd gr, ccording to Greene et l (1976) nd s modified y Hoffmnn-Cmpo et l (1985). The rutin used in the

2 Septemer - Octoer 21 Neotropicl Entomology 39(5) 775 experiments ws purchsed from Sigm, nd the mount used ws sed on the percentge of flvonoid oserved y Hoffmnn-Cmpo et l (21) in insect-resistnt soyen genotypes, which ws.641% (dry weight) of rutin in the polr frction of the folir extrct of PI Biologicl ctivity of rutin-supplemented csein-sed rtificil diets on A. gemmtlis. Three experiments were crried out to evlute the effects of different concentrtions of csein nd rutin on the iology of A. gemmtlis. In experiment 1, tretments consisted on stndrd diet (14 g of csein) without rutin (here termed ) or with.65% of rutin (+.65%R); diet with hlf of the usul csein quntity (7gC), without or with.65% rutin (7gC+.65%R); nd diet with no csein (gc) nd without or with.65% rutin (gc+.65%r). Experiment 2 ws similr to experiment 1, ut the rutin concentrtion ws incresed to 1.3%. Finlly, in the third experiment, stndrd diets with 14 g of csein () nd with or without two concentrtions of rutin,.65% (+.65%R) nd 1.3% (+1.3%R), were tested. These were compred to diets without csein (gc), without csein plus.65% rutin (gc+.65%r), nd without csein plus 1.3% rutin (gc+1.3%r). Extrction of flvonoids from soyen proteins. The rutin from soyen proteins used in prepring the diets ws extrcted y using 8% methnol (MeOH) in proportion of 1:1 (v:w) under continuous gittion (1 rpm) for pproximtely 18 h. The extrct otined ws filtered in Buchner funnel with Frmex quntittive fst filtrtion pper, nd the extrction process ws conducted three times to gurntee complete extrction of the flvonoids. Before the diets were prepred, ll the solvent (MeOH) ws completely evported in n ir chmer. Experiment setup. The rtificil diet ws prepred s descried y Hoffmnn-Cmpo et l (1985). Rutin ws dissolved in distilled wter nd mixed with the rtificil diet t temperture elow 5ºC. Lte second lrvl instrs kept on their respective tretment-diets since htching, were plced individully in smll crylic cups with sterilized crdord lids, nd mintined under controlled conditions (25 ± 2ºC; 7 ± 1% UR; 12-h photoperiod). Lrve were inspected dily, nd the mortlity nd the development time of ech stge were recorded. To estimte the dry weight of the diets, five cups were tken from ech tretment, weighed, dried in n oven until constnt weight (6 o C, for 72h), nd re-weighed. The correction fctor for fresh/dry weight of ech diet ws clculted, nd the vlues otined were multiplied y the fresh weight of food contined in ech cup. To vlidte the correction fctor for fresh/dry weight, nlyses of liner regression nd correltion were crried out. For the three sets of experiments, the equtions were: Ŷ 1 = X 1 (R 2 =.71) (experiment 1), Ŷ 2 = X 2 (R 2 =.94) (experiment 2), nd Ŷ 3 = X 3 (R 2 =.9) (experiment 3). The food remining in the cups fter lrvl consumption ws seprted from the feces nd oth were oven-dried, s descried ove, nd weighed. To otin the mount of food consumed y the lrve, the dry weight of the non-consumed food ws sutrcted from the initil dry weight of the food. At 48h fter puption, pupe were weighed, killed, nd dried until constnt weight to determine their dry weights. Sttisticl nlyses. A fully rndomized lock design ws used for ll experiments. The first nd third experiments hd 65 replictes ech, nd the second hd 6 replictes. Lrvl mortlity during ech instr, totl lrvl mortlity, pupe weight (dry, in mg), totl feeding time (dys), food consumed (mg), nd feces weight (mg) were evluted. The mortlity ws compred y using the chi-squre test (χ 2 ), ccording to Bnztto & Kronk (1992), t 5% proility level. The remining vriles were nlyzed in three steps. In the first step, sttisticl tests were used to confirm if the dt showed ll the fundmentls for the nlysis of vrince (ANOVA), followed y Tukey nd Krmer tests for comprison of tretment mens, t 5% proility level. In the lst step, the nlysis of covrince (ANCOVA) ws crried out to estimte growth, food consumption, nd the efficiency of ssimiltion nd conversion of ingested nd digested food into iomss, s proposed y Ruenheimer & Simpson (1992). Insect growth nd food consumption in the different tretments were otined y djusting, respectively, pupl weight nd food consumption for the covrite feeding time. The efficiency in the conversion of ingested nd digested food into iomss ws clculted y the djustment of pupl weight, respectively, for the food consumed nd digested (the mount of food consumed minus the mount of feces produced y the lrve). The food ssimiltion ws otined fter djusting the mount of feces produced for the covrite mount of food consumed. After the ANCOVA, whenever the interction etween the covrite nd the tretment ws significnt, the effect of the tretments djusted for the covrite ws included. However, when the interction (covrite x tretment) ws not significnt, the prllel-lines model ws used, considering only the effect of the tretments. All sttisticl nlyses were performed using the SAS - Sttisticl Anlysis System sttisticl pckge (SAS Institute 1996). Results Experiment 1. Mortlity, feeding time, pupl weight, food consumption, nd feces weight of A. gemmtlis. Most of the tretments cused some mortlity. The highest mortlity (χ 2 = 51.86, P >.5) ws oserved for lrve fed diet contining 7gC +.65%R (38.29%), followed y those fed gc +.65%R (26.15%), +.65%R (1.77%), nd nd gc (1.54%). No mortlity ws oserved on insects fed the 7gC diet. A significnt effect of the tretments on pupe weight, food consumption, feces weight, nd feeding time ws oserved (Tle 1). The weight of the pupe whose lrve te the gc diet did not differ from pupe from lrve fed the nd 7gC diets. The lowest pupl weights nd longest feeding times were oserved when lrve were fed on diets with rutin (gc+.65%r, 7gC+.65%R, nd +.65%R). The highest consumption ws oserved in

3 776 Slvdor et l - Do Different Csein Concentrtions Increse the Adverse Effect of Rutin on the Biology of... Tle 1 Mens (±SE) of dry weight of pupe, consumed diet, feces nd feeding time of Anticrsi gemmtlis fed on rtificil diet contining different concentrtions of csein (C) plus,65% rutin (R). Diet 1 Mens followed y the sme letter on the column re not significntly different y Tukey nd Krmer test, t 5% proility; *** P <.1. Weight (mg) Pupe Consumed diet Feces Feeding time (dys) ± ± ± ±.22 c +R ± ± 7.83 c ± 4.43 c ±.22 7gC ± ± ± 4.28 c 9.58 ±.21 c 7gC+R 39.5 ± ± 9.9 c ± 5.6 c 14.3 ±.28 gc ± ± 7.5 c ± ±.21 c gc+r 42. ± ± 9.4 c ± 5.11 c 14.2 ±.25 F Vlues 24.64*** 1.3*** 5.9*** 82.31*** lrve fed on rutin-free csein-sed diets (7gC nd ), when compred to the other tretments. Lrve fed the gc diet produced the most feces. Growth, food consumption, conversion efficiency of ingested nd digested food into iomss, nd ssimiltion of food of A. gemmtlis fed rtificil diets with different concentrtions of csein nd.65% rutin. The ANCOVA indicted significnt nd interctive effect etween tretments nd the covrites feeding time (Tle 2), food consumption (Tle 2c), nd food digested (Tle 2d), regrding pupl weight. There ws lso n interction etween tretments nd the covrite food consumption, regrding feces weight (Tle 2c). There ws no significnt interctive effect etween tretments nd feeding time, regrding food consumption (Tle 2); consequently, this reltionship cn e represented y the prllel-lines model (Figs 1). The extension in lrvl feeding time (covrite) did not result in n increse in pupl weight, considering the negtive or stle trends of the curves tht represent the dt otined (Fig 2). A negtive reltionship etween food consumption Tle 2 Anlysis of covrince (ANCOVA) djusting Anticrsi gemmtlis pupe weight nd consumed diet y covrite feeding time (, ); pupe weight nd feces weight y covrite consumed diet (c); nd pupe weight y covrite digested food (d). Vrition GL NS Non significnt; *P <.5; **P <.1; *** P <.1 F vlue Pupe weight Consumed diet Feces weight () Feeding time (covrite) *** 8.43** - Tretment **.61 NS - Feeding time x tretment *.71 NS - Residul () Tretment ** - Feeding time ** - Residul (c) Consumed diet (covrite) *** *** Tretment NS NS Consumed diet x tretment ** ** Residul (d) Digested diet (covrite) 1.71 NS - - Tretment *** - - Digested diet x tretment ** - - Residul

4 Septemer - Octoer 21 Neotropicl Entomology 39(5) c Pupl weight (mg) Pupl weight (mg) Feeding time (dys) +R 7gC 7gC+R gc gc+r Feces weight (mg) d +R 7gC 7gC+R gc gc+r Fig 1 Reltionship etween feeding time (dys) nd pupl weight (mg) () or consumption (mg) (), nd reltionship etween consumption (mg) nd pupl weight (mg) (c) or feces weight (mg) (d) of Anticrsi gemmtlis fed on rtificil diet contining different concentrtions of csein plus.65% rutin. Inserted grph shows ANCOVA mens. (For sttisticl nlysis see Tle 2). nd insect feeding time (Fig 2) ws lso oserved; lrve tht extended their feeding time did not consume more food. In Fig 2, it is possile to oserve the occurrence of quntittive fctor (Horton & Redk 1996) ffecting lrve fed on the diet with rutin. The lest-squres men (grph insert, Fig 2) indictes tht the lrve fed the gc diet (without csein nd without csein plus rutin) consumed less food thn those fed on the 7gC diet without rutin. Lrve fed on diets with rutin, 7gC+.65%R, +.65%R, nd gc+.65%r, were less efficient in converting the ingested food into iomss (pupl weight djusted for the covrite consumption) (Fig 1c). The increse in consumption did not result in n increse in pupl weight. In the evlution of food ssimiltion (Fig 1d), feces production ws dependent on the different tretments, s well s on the quntity of ingested food. The dt indicte positive reltionship etween the response vrile (weight of feces) nd the covrite (consumption); lrve tht consumed more food lso produced more feces. Lrve fed on diets with rutin, independently of csein concentrtion, were less efficient in converting the food digested [pupe weight djusted y the covrite food digested (consumption feces)] into iomss (Fig 2), s suggested y the lower inflections oserved in the curves tht represent these tretments. Experiment 2: Mortlity, feeding time, pup weight, food consumption, nd feces weight of A. gemmtlis. Similrly to the results of the previous experiment, the highest mortlity rte (χ 2 = 134,2, P > 5) occurred for the lrve fed the 7gC+1.3% R (64.98%) diet, followed y those fed the gc+ 1.3%R (53.32%), + 1.3%R (49.98%), nd 7gC (1.66%) diets. All the lrve fed the nd gc diets survived. The feeding time of the lrve ws shorter for insects fed the diet without rutin, nd longer for those fed the diets with rutin, 7gC+1.3%R, gc+1.3%r, nd +1.3%R (Tle 3). Lrve fed the +1.3% nd gc+1.3%r diets hd lower pupl weights when compred to those fed rutin-free csein-sed diets (7gC nd ). The highest consumption ws oserved for insects fed the diet nd the lowest on the gc diet; no sttisticlly significnt differences were oserved mong the other tretments (+1.3%R, 7gC, 7gC+1.3%R, gc+1.3%r). The lowest mount of feces ws oserved in the gc tretment, differing from diets contining csein ut without rutin ( nd 7gC).

5 778 Slvdor et l - Do Different Csein Concentrtions Increse the Adverse Effect of Rutin on the Biology of... Pupl weight (mg) Digested food (mg) Feeding time (dys) +R 7gC 7gC+R gc gc+r c +R 7gC c 7gC+R c gc c gc+r Fig 2 Reltionship etween weight of digested food (mg) nd pupl weight nd of Anticrsi gemmtlis fed on rtificil diet contining different concentrtions of csein plus.65% rutin (), nd reltionship etween feeding time (dys) nd consumption (mg) of A. gemmtlis fed on rtificil diet contining different concentrtions of csein plus 1.3% rutin (). Inserted grph shows ANCOVA mens. (For sttisticl nlysis see Tles 2 nd 4, respectively). Growth, food consumption, conversion efficiency of ingested nd digested food into iomss, nd ssimiltion of food of A. gemmtlis fed rtificil diets with different concentrtions of csein nd 1.3% rutin. The ANCOVA indicted tht there ws no interction etween tretments nd the covrite feeding time, regrding either pupl weight or diet consumed (Tle 4). Also, there were no interctions etween tretments nd the covrites food consumption (Tle 4c), or food digested (Tle 4e) in their reltionship to pup weight. Therefore, the interctive effect ws oserved only etween tretments nd the covrite food consumption, regrding feces weight (Tle 4c). The insects consumption ws ffected y the tretments (Tle 4), with no effect of feeding time (covrite). The nlysis of the curves tendency in Fig 2 showed tht the lrve fed on the diets contining rutin hd n extended feeding time without incresing their consumption. Lrve fed the diet consumed the lrgest mount of food, followed y those fed the 7gC diet. The lowest consumption ws oserved for lrve fed the gc diet (grph insert, Fig 2). The conversion efficiency of the food ingested (pup weight x food consumption covrite) showed positive reltionship (Fig 3); the more food ws consumed y the lrve, the higher ws the pupe weight, with those fed the diets without csein nd rutin eing more efficient in the conversion of ingested food when compred to those fed stndrd diet with ( + R) or without rutin () (grph insert, Fig 3). The insects fed the C or 7gC diets, ut without rutin, converted more of the food ingested into iomss thn those fed the 14g + R diet. The interction etween the food consumption covrite nd feces weight suggested tht the production of feces depended on the tretment, ut lso on the quntity of food consumed (Fig 3). The tendency curves of the dt indicte positive reltionship; in other words, the Tle 3 Mens (±SE) of dry weight of pupe, consumed diet, feces nd feeding time of Anticrsi gemmtlis fed on rtificil diet contining different concentrtions of csein (C) plus 1.3% rutin (R). Diet Weight (mg) Pupe Consumed diet Feces Feeding time (dys) ± ± ± ±.27 c +R ± ± ± ±.37 7gC ± ± ± ±.27 c 7gC+R ± ± c ± ±.49 gc ± ± 6.32 c ± ±.26 c gc+r 46.4 ± ± 9.62 c ± ±.4 F Vlues 4.63*** 43.6*** 8.48*** *** Mens followed y the sme letter on the column re not significntly different y Tukey nd Krmer, t 5% proility; *** P <.1.

6 Septemer - Octoer 21 Neotropicl Entomology 39(5) 779 Tle 4 Anlysis of covrince (ANCOVA) djusting Anticrsi gemmtlis pupe weight nd consumed diet y covrites feeding time (, ); pupe weight nd feces weight y covrite consumed diet (c, d); nd pupe weight y covrite digested food (e, f). Vrition GL NS Non significnt; * P <.5; **P <.1; *** P <.1 F vlue Pupe weight Consumed diet Feces weight () Feeding time (covrite) 1.7 NS.3 NS - Tretment 5.17 NS 1.89 NS - Feeding time x tretments 5.37 NS.95 NS - Residul () Tretment NS 4.23*** - Feeding time 1.8 NS.3 NS - Residul (c) Consumed diet (covrite) *** *** Tretment 5.74 NS - 8.3*** Consumed diet x tretment 5.64 NS *** Residul (d) Tretment *** - - Consumed diet *** - - Residul (e) Digested food (covrite) *** - - Tretment NS - - Digested food x tretment 5.98 NS - - Residul (f) Tretment ** - - Digested food *** - - Residul higher the consumption, the more feces were produced. However, the tendency curve of the dt for the insects fed the diet showed lower inflection, suggesting higher food ssimiltion, considering tht even with incresed consumption, the lrve produced reltively less feces, proly retining more food. The conversion of food digested into iomss (pup weight x food digested covrite) (Fig 4) followed the sme tendency oserved in the conversion of ingested food (Fig 3), with lrve incresing the mount of food digested nd producing hevier pupe. The insects fed the 7gC nd gc diets were most efficient in converting digested food into iomss (grph insert, Fig 4). Experiment 3: Mortlity, feeding time, pupe weight, food consumption, nd feces weight of A. gemmtlis. According to the χ 2 test (P >.5), the tretments cused some mortlity. Lrve fed the gc + 1.3%R diet showed the highest mortlity rte (6%), followed y the + 1.3R (43.7%), gc+.65r (27.7%), nd +.65R (18.46%) diets. Mortlity rtes of lrve fed the diets without rutin ( nd gc) were 1.54% nd 7.7%, respectively. The highest mortlity percentges were oserved during third instr for oth tretments. Tle 5 shows tht the gc + 1.3%R tretment yielded pupe with the lowest men weight, differing from the other tretments; the lrvl weight in the stndrd diet () ws the highest. In this tretment, higher consumption nd shorter feeding time compred to the other tretments were lso oserved; lrve fed on diets without csein showed lower consumption nd lower feces weight. The longest feeding time ws oserved in insects fed the gc + 1.3%R diet (15. ±.55 dys). Growth, food consumption, conversion efficiency of ingested nd digested food into iomss, nd food ssimiltion of A. gemmtlis fed n rtificil diet with different concentrtions of csein nd rutin. An interctive effect (evluted y ANCOVA) etween tretment nd food consumption covrite ws oserved, in reltion to the mount of feces (Tle 6c). However, there ws no interctive effect etween tretment nd

7 78 Slvdor et l - Do Different Csein Concentrtions Increse the Adverse Effect of Rutin on the Biology of Pupl weight (mg) R 7gC 7gC+R gc gc+r Pupl weight (mg) Digested food (mg) R 7gC 7gC+R gc gc+r +R 7gC 7gC+R gc gc+r Feces weight (mg) R 7gC 7gC+R gc gc+r Fig 3 Reltionship etween consumption (mg) nd pupl weight (mg) () or feces weight (mg) () of A. gemmtlis fed on rtificil diet contining different concentrtions of csein plus 1.3% rutin Inserted grph shows ANCOVA mens. (For sttisticl nlysis see Tle 4). the feeding time covrite (Tle 6), in its reltionship to pupe weight nd to food consumption. Additionlly, no interction etween tretment nd food consumption (covrite), in the conversion of ingested food into iomss evluted y the reltionship consumption x pup weight (Tle 6c), nor of conversion of digested food into iomss (digested food x pup weight, Tle 6e) ws detected. As, in this lst cse, there ws no significnt effect of the covrite (Tle 4f), the dt otined y ANOVA were ccepted s vlid. The insects consumption (Fig 4) ws negtively relted to feeding time; in other words, those insects fed for longer period ( +.65%R, + 1.3%R, gc +.65%R, nd gc + 1.3%R) did not show proportionl increse in the quntity of food consumed. The ANCOVA men (grph insert, Fig 4) indictes tht the insects fed stndrd diet () consumed more when compred to the other tretments. In the evlution of the conversion efficiency of Feeding time (dys) +.65R +1.3R gc gc+.65r gc+1.3r Fig 4 Reltionship etween weight of digested food (mg) nd pupl weight (mg) of A. gemmtlis fed on rtificil diet contining different concentrtions of csein plus 1.3% rutin (), nd reltionship etween feeding time (dys) nd consumption (mg) of A. gemmtlis fed on rtificil diet contining different concentrtions of csein plus plus.65% nd 1.3% rutin (). Inserted grph shows ANCOVA mens. (For sttisticl nlysis see Tles 4 e 6, respectively). ingested food y ANCOVA (Fig 5), positive reltionship etween the fctors ws oserved; the more the lrve consumed, the higher the pupe weight. Insects fed the stndrd diet () were most efficient in converting food into iomss; the lest efficient insects were those fed the gc+1.3%r diet, followed y the + 1.3%R diet (grph insert, Fig 5). The food consumption (covrite) nd the tretments significntly ffected the feces weight; the mount of feces produced y the insects ws dependent on n interctive effect etween the covrite nd tretments. In the dispersion grph (Fig 5), gin positive reltionship etween the 14g+R,65 14g +R1,3 c gc c c gc+r,65 gc+r1,3

8 Septemer - Octoer 21 Neotropicl Entomology 39(5) 781 Tle 5 Mens (±SE) of dry weight of pupe, consumed diet, feces nd feeding time of Anticrsi gemmtlis fed on rtificil diet contining different concentrtions of csein (C) nd.65% nd 1.3% rutin (R). Diet Weight (mg) Pup Consumed diet Feces weight Feeding time (dys) ± ± ± ±.36 d 14g+.65%R 46.2 ±.1 c ± ± 4.63 c 14.6 ±.4 14g+1.3%R ± 1.24 c ± ± ±.5 c gc 5.9 ± ± 5.84 c ± 4.22 c ±.37 c gc+.65%r 44.5 ± 1.11 c ± 7.11 c ± 5.14 c ±.45 c gc+1.3%r ± 1.36 d ± 8.72 c ± 6.3 c 15.4 ±.55 F vlue 54.65*** 36.57*** 7.74*** 26.34*** Mens followed y the sme letter on the column re not significntly different y Tukey nd Krmer, t 5% proility; *** P <.1. fctors could e oserved. However, the tendency curve of the grph indictes tht the insects fed stndrd diet () retined more food y producing smller mounts of feces, even when they incresed their consumption. Therefore, they were more efficient in ssimilting food, which proly explins their etter overll performnce. Tel 6 Anlysis of covrince (ANCOVA) djusting Anticrsi gemmtlis pupe weight nd consumed diet y covrites feeding time (, ); pupe weight nd feces weight y covrite consumed diet (c, d); nd pupe weight y covrite digested food (e, f). Vrition GL NS Non significnt; *P <.5; **P <.1; *** P <.1 F vlue Pupe weight Consumed diet Feces weight () Feeding time (covrite) 1.1 NS 6.94** - Tretment 5 5.1*** 3.1** - Feeding time x tretment NS 1.44 NS - Residul () Tretment *** 27.33*** - Feeding time 1.1 NS 6.88** - Residul (c) Consumed diet (covrite) ** *** Tretment 5.71 NS NS Consumed diet x tretment NS * Residul (d) Tretment *** - - Consumed diet ** - - Residul (e) Digested food (covrite) NS - - Tretment * - - Digested food x tretment NS - - Residul (f) Tretment *** - - Digested food NS - - Residul

9 782 Slvdor et l - Do Different Csein Concentrtions Increse the Adverse Effect of Rutin on the Biology of... Pupl weight (mg) Feces weight (mg) Fig 5 Reltionship etween consumption (mg) nd pupl weight (mg) () or feces weight (mg) () of A. gemmtlis fed on rtificil diet contining deferent concentrtions of csein plus.65% nd 1.3% rutin. Inserted grph shows ANCOVA mens. (For sttisticl nlysis see Tle 6) c +.65R cd R gc +.65R +1.3R gc gc+.65r Discussion gc+1.3r The highest mortlity rtes were oserved for insects fed diets with hlf the concentrtion of csein in reltion to the stndrd diet used for mss rering of A. gemmtlis, or without dded csein, ut contining rutin. According to Stmp (1994), flvonoids generlly ffect defoliting insects minly during instr chnges nd A. gemmtlis showed n ccentuted mortlity rte during their moulting to the lst instr. During this stge, insects generlly increse food consumption nd will, consequently, ingest higher mount of llelochemicls. Flvonoids incorported to rtificil diets lso cused higher mortlity of sixth instrs of A. gemmtlis (Gzzoni et l 1997). In this prticulr cse, flvnoids were suggested to lter the ctivity of enzymes nd hormones, locking iochemicl pthwys nd reducing the ssimiltion of essentil sustnces nd nutrient storge. The prolongtion of the feeding time of A. gemmtlis c gc+,65r d gc+1.3r lrve fed on rutin-diet my hve een tenttive to uptke the required nutrients to enhnce survivl. However, A. gemmtlis fed diets with different rutin concentrtions in comprison to rutin-free diets were lso shown to hve reduced food consumption (Hoffmnn-Cmpo et l 26, Piuelli et l 26), indicting the lrv ttempted to void poisoning y eting smller mels, for prolonged time, to otin the idel weight for puption. Although this strtegy llows them to survive, the prolonged lrvl development will certinly mke them lrgely prone to the ttck of their nturl enemies in field conditions, incresing their mortlity risks. In generl, our results showed tht lrve fed diet contining rutin in ny concentrtion consumed less food nd were less efficient in converting food to iomss, s ws lso the cse for lrve on csein-free diet without dded rutin. During choice-tests with Schistocerc mericn, Bernys & Ruenheimer (1991) oserved tht insects on low-protein diets supplemented with rutin fed smller portions of food for longer periods, while in long term experiments, rutin negtively ffected not only S. mericn growth, ut lso the conversion of ingested food to iomss (Bernys et l 1991), s demonstrted in here for A. gemmtlis. The lower consumption of the cterpillrs in tretments without csein is proly due to the lck of phgostimultory effects ttriuted of this protein (Prr 1979), s oserved for Ostrini nuillis (Beck 1956). A shorter feeding time, higher lrvl nd/or pupl weight, nd lower mortlity rtes re prmeters tht indicte n dequte growth rte for insects (Soo Hoo & Frenkel 1966). Anticrsi gemmtlis fed on diets contining rutin, especilly on the 7gC nd gc csein diets, did not show dequte growth rtes compred to insects fed stndrd diets with rutin. Furthermore, the effect of the flvonoid ecme more pronounced only when insects were fed n unlnced diet (with low concentrtion or lck of csein). Our results did not corroorte those of Duffey & Stout (1996), who stted tht high-energy proteins such s csein cn ccentute the negtive effects of phenolic compounds (chlorogenic cids nd rutin) to lepidoptern lrve. Considering the results otined so fr, we cn suggest tht with n dequte concentrtion of csein, there is no interctive effect of this protein with the flvonoid rutin on the iology of the velveten cterpillr, nd thus, csein does not increse the dverse effects of rutin on A. gemmtlis lrve. Acknowledgments To Sérgio Henrique d Silv for his ssistnce in the lortory experiments. To Dr Normn Neumier for reding n erlier version of the present pper. To Cpes for providing scholrship to MCS. This pper ws pproved for puliction y the Editoril Bord of Emrp Soj, s mnuscript 2/28. References Bnztto D A, Kronk S N (1992) Experimentção grícol. Funep, Joticl, 247p.

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