Growth and nutritional physiology of Spodoptera frugiperda (Lepidoptera: Noctuidae) fed on Meliaceae fractions

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1 Revist Colomin de Entomologí 41 (1): (Enero - Junio 2015) 33 Growth nd nutritionl physiology of Spodopter frugiperd (Lepidopter: Noctuide) fed on Melicee frctions Crecimiento y fisiologí nutricionl de Spodopter frugiperd (Lepidopter: Noctuide) limentds con frcciones de Melicee ANGELINA MARIA MARCOMINI GIONGO 1, JOSÉ DJAIR VENDRAMIM 2, SÂMYA DANIELLE LIMA DE FREITAS 3 nd MARIA FÁTIMA DAS GRAÇAS FERNANDES DA SILVA 4 Astrct: The effect of hexne, dichloromethne, ethyl cette nd hydrolcoholic frctions otined from ethnolic extrct of Trichili pllid, T. pllens nd Toon cilit on growth nd nutritionl physiology of Spodopter frugiperd ws evluted. First instr lrve were fed on diet treted with frctions for ten dys. The most ctive frctions were pplied on the diet nd offered to lrve until the pupl stge to verify effects on growth nd survivl. To clculte the nutritionl indices, frctions were incorported into the diet offered to fourth instr lrve for four dys. The dichloromethne frction of T. pllens stems ffected growth nd survivl of S. frugiperd lrve, nd frctions of T. pllid leves nd stems, nd of To. cilit leves nd fruits ffected lrvl growth. Nutritionl nlysis indicted tht the frction of To. cilit leves cts minly s phgodeterrent, while dichlorometne frctions of T. pllid stems nd leves, To. cilit fruits nd T. pllens stems cuse toxic effects fter ingestion, reducing the efficiency of food conversion nd incresing the metolic cost to the lrve. Key words: Insect metolism. Plnt extrcts. Sulethl effects. Resumen: Se evluó el efecto de frcciones de hexno, diclorometno, cetto de etilo e hidrolcohólic, otenids prtir del extrcto de etnol de Trichili pllid, Trichili pllens y Toon cilit sore el crecimiento y fisiologí nutricionl de Spodopter frugiperd. Lrvs de primer estdio se limentron con diet trtd con dichs frcciones durnte diez dís. Ls frcciones más ctivs fueron plicds sore l diet y se ofrecieron ls lrvs hst pup pr verificr efectos sore el crecimiento y sorevivenci. Pr clculr los índices nutricionles, l diet con ls frcciones fueron ofrecids ls lrvs de curto estdio durnte cutro dís. L frcción de diclorometno de rms de T. pllens fectó el crecimiento y l sorevivenci de ls lrvs. Por otr prte, ls frcciones de diclorometno de rms y hojs de T. pllid y hojs y frutos de To. cilit fectron el crecimiento lrvl. El nálisis nutricionl indicó que l frcción de hojs de To. cilit ctú principlmente como disusor de limentción mientrs que ls frcciones de diclorometno de rms y hojs de T. pllens, fruts de To. cilit y rms de T. pllens cusn efectos tóxicos después de l ingestión, reduciendo l eficienci de conversión de limento y umentndo el costo metólico pr ls lrvs. Plrs clve: Índices nutricionles. Fgodisusión. Introduction One of the chllenges for insect control is the development of more effective strtegies thn chemicl insecticides, s there re insect popultions resistnt to most synthetics currently used, nd lso sfer for mn, the environment nd nturl enemies (Rttn 2010). Botnicl insecticides re suggested s n lterntive to synthetics (Akhtr et l. 2012), nd the fmily Melicee is currently well investigted due to the occurrence of limonoid triterpenes, promising compounds tht hve iologicl ctivities on insects like insecticidl, phgodeterrent nd growth regulting (Ismn et l. 2002; Roy nd Srf 2006). Most work hs een focused on the neem tree Azdircht indic A. Juss. nd the limonoid zdirchtin (Ntlli nd Menkissoglu-Spiroudi 2011). Other ioctive compounds re ville on gener Cedrel, Khy, Trichili, Chisocheton, Toon nd Tureu (Ismn 1995; Adelgleil et l. 2001). There re reports of ioctivity of other Melicee like Trichili pllid Swrtz, Trichili pllens C. DC. nd Toon cilit M. Roemer extrcts on some insect pests (Bezerr- Silv et l. 2010; Cunh et l. 2005; Pvrini et l. 2010), including Spodopter frugiperd (J. E. Smith) (Lepidopter: Noctuide) (Rodríguez nd Vendrmim 1997; Roel et l. 2000; Bogorni nd Vendrmim ). However there re no studies out frctiontion of these extrcts to evlute the effect of the frctions on the fll rmyworm. Considering the potentil of these three Melicee for the control of insects, this study ws crried to evlute the effect of the frctions of ethnolic extrcts on the growth, survivl nd nutritionl physiology of S. frugiperd. Mterils nd methods The chemicl procedures were crried out t the Lortório de Produtos Nturis of Universidde Federl de São Crlos (UFSCr), nd iologicl tests with S. frugiperd t Lortório de Plnts Inseticids of Escol de Superior de Agricultur Luiz de Queiroz (ESALQ), in São Pulo, Brzil, from Octoer 2010 to Decemer Insects rering. S. frugiperd ws mintined in lortory under controlled conditions (25 ± 2 C, RH = 60 ± 10% nd 14-hour photoperiod). Lrve were fed on n r- ¹ Ph. D. in Entomology. Escol Superior de Agricultur Luiz de Queiroz, Av. Pádu Dis, 11, , Pircic, Brzil. mmrcomini@yhoo.com.r. Corresponding uthor. ² Ph. D. in Entomology nd Professor. Escol Superior de Agricultur Luiz de Queiroz, Av. Pádu Dis, 11, , Pircic, Brzil. ³ M. Sc. in Chemistry. Universidde Federl de São Crlos, Rodovi Wshington Luiz, km 235, São Crlos, Brzil. 4 Post Doctor in Chemistry nd Professor. Universidde Federl de São Crlos, Rodovi Wshington Luiz, km 235, São Crlos, Brzil.

2 34 Revist Colomin de Entomologí Angelin Mri Mrcomini Giongo et l. tificil diet (Greene et l. 1976) nd dults received honey solution 1%. Plnt mteril. Stems nd leves of T. pllid (voucher ESA 81288) were collected in Octoer 2010, nd stems, leves nd fruits of To. cilit (voucher ESA 78420) in Jnury 2011, oth from the ESALQ cmpus. Stems nd leves of T. pllens (voucher ESA 81286) were collected in April 2011 from Ljedo ds Orquídes in Spopem-PR. The mteril ws dried in drying oven with ir circultion t 40 C, ground in Wiley mill nd stored in glss vils. Extrcts nd frctions. Ethnolic extrcts were prepred using n Ik Ultr Turrx (T25) dispersing instrument. Five extrctions of five minutes ech t 8,000 rpm nd room temperture were crried out, y dding 1 L of ethnol per 200 g of powdered plnt mteril. The extrcts were filtered through filter pper nd the solvent ws eliminted under vcuum on rotry evportor (45 C) until dry. Ech residue of ethnol extrct (10 g) ws dissolved in methnol (100 ml) nd wter (300 ml) nd exhustively extrcted y consecutive liquid/liquid prtition with solvents of incresing polrity (hexne, dichloromethne nd ethyl cette) using seprting funnel, which ws shken to mix the solvent with the extrct nd then llowed to stnd for phse seprtion. The hexne, dichloromethne, ethyl cette nd lst remining hydrolcoholic frctions were evported under vcuum. At the end of the process, the following were otined: hexne, dichloromethne, ethyl-cette nd hydrolcoholic frctions of T. pllid leves, T. pllid stems, T. pllens leves, T. pllens stems, To. cilit leves, To. cilit stems nd To. cilit fruits, totling 28 frctions. Bioctivity ssys. Frctions were evluted in four iossys: 1) ll frctions of T. pllid; 2) frctions of T. pllens stems; 3) frctions of T. pllens leves; nd 4) ll frctions of To. cilit. Hexne, dichloromethne nd ethyl cette frctions of three plnts were redissolved in cetone nd wter (1:1) nd hydrolcoholic frctions were redissolved in ethnol nd wter (1:1) t 1% (10,000 ppm) (weight y volume). The rtificil diet ws plced in multi-well pltes (2 ml per well) nd 30 µl of frctions t 1% were pplied on the diet surfce. After two hours for solvent evportion, first instr lrve (24 h of ge) were plced one per well. Three controls were used: wter nd cetone (1:1), wter nd ethnol (1:1), nd only wter to evlute the effect of the solvents. Ech tretment consisted of four repetitions of 24 lrve, except in the ssy with frctions of T. pllens leves, which consisted of five replictes of twelve lrve. Mortlity ws recorded dily, nd fter ten dys the surviving lrve were weighed. Effect on lrvl growth nd survivl. In second test, the most ctive frctions were selected to determine the effects on lrve growth nd survivl. Frctions dissolved t 1% were pplied on the diet surfce in glss tues ( cm) contining 3 ml of diet (40 µl/tue), nd first instr lrve were individulized in tues. Four replictes of five lrve ech were performed. The prmeters evluted were mortlity, durtion of lrvl stge nd pupl weight t 24 hours. Effect on nutritionl physiology. To understnd the effects of the most ctive frctions on the nutritionl physiology of S. frugiperd, nutritionl indices of fourth instr lrve tht ingested treted diet were clculted. Frctions were dissolved in cetone nd incorported into the rtificil diet t concentrtion of 1,000 ppm (1 mg/g diet). The rtificil diet contining the frctions or cetone (control) ws plced in glss tues of known weight, which were weighed gin to otin the initil weight of diet. Fourth instr lrve were weighed nd individulized in the tues. Initil fresh nd dry weights of n liquot of the sme instr lrve were determined. A diet contining only cetone ws used s control. Twenty fourth instr lrve were used per tretment (independent replictes), nd fter four dys of feeding, lrve nd feces were seprted from the remining diet. Lrve were killed y freezing nd ll the mteril ws plced in drying oven t 50 C for seven dys, to otin the dry weight, which ws used for the determintion of nutritionl indices. From these dt the weights were otined: food supplied, food remining, food ingested (I), feces (F), lrve weight gin (B) nd lrve men weight (C). Then the following indices were clculted in the durtion of feeding time (T): the reltive consumption rte (RCR = I / (C T)), the reltive metolic rte (RMR = M / (C T)), reltive growth rte (RGR = B / (C T)), the efficiency of conversion of ingested food (ECI = (B / I) 100) nd digested food (ECD = (B / (I - F)) 100), the pproximte digestiility (AD = ((I - F) / I) 100), the metolic cost (MC = ECD), weight of the ssimilted food (I - F) nd weight of the metolized food (M = (I - F) - B) (Scrier nd Slnsky Jr. 1981). Dt nlysis. All iossys were crried out in completely rndomized design. The nutritionl indices were sujected to covrince nlysis (Ruenheimer nd Simpson 1992), in which the denomintor ws used s covrite for ech index. Mens were compred y the Tukey-Krmer test (p 0.05), with the sttisticl progrm SAS 9.2 (SAS Sttisticl Anlysis System, 2009). For the other nlyses, the sttisticl progrm R version (R Development Core Tem, 2013) ws used. Dt with inomil distriution (mortlity) were nlyzed y GLM (Generlized Liner Models) y cind procedure using the chi-squre test (p 0.05) for comprison of mens. The other dt were sujected to Brtlett s test to check homogeneity of vrinces, nd to the Shpiro-Wilk test to verify the normlity of residuls. Then n nlysis of vrince ws done (F test, p 0.05), nd mens were compred y the Tukey test (p 0.05), using the glm nd glht procedures. Results nd discussion Bioctivity ssys. Among hexne, dichloromethne, ethyl cette nd hydrolcoholic frctions of T. pllid leves nd stems pplied on the diet offered to the lrve of S. frugiperd (Tle 1), the dichloromethne frction of stems ws the one tht cused higher lrvl mortlity thn those recorded in the ethnol, cetone nd wter controls. For oth stems nd leves in ethyl cette, hexne nd hydrolcoholic frctions, mortlity did not differ from controls. There ws no difference etween the wter control nd the ethnol nd cetone controls (solvents used to dissolve the frctions), indicting no effect of them on the lrve. Despite the low mortlity levels, the weight of lrve tht ingested the diet contining dichloromethne frctions of T. pllid leves nd

3 Melicee frctions ginst Spodopter frugiperd 35 stems ws reduced fter ten dys, not reching 10% of those recorded in the other tretments. Similr results were oserved with the frctions of T. pllens stems, where only the dichloromethne frction cused lrvl mortlity higher thn in the three controls, nd drsticlly ffected lrvl growth, which reched mximum of 1.5% of the weight recorded in ll tretments including controls (Tle 1). For ethyl cette, hexne nd hydrolcoholic frctions, lrvl mortlity did not differ from the controls. There ws no difference etween tretments with frctions of T. pllens leves nd controls in reltion to lrvl mortlity. For lrvl weight, the vlue otined in the hexne frction ws lower thn tht otined in control wter, ut did not differ from ethnol nd cetone controls (Tle 1). Frctions of To. cilit stems, leves nd fruits did not reduce lrvl survivl, ut dichloromethne frctions of leves nd fruits ffected the lrve development (Tle 2). Bioctivity of these Melicee extrcts ginst S. frugiperd ws lredy reported (Rodríguez nd Vendrmim 1997; Roel et l. 2000; Torrecills nd Vendrmim 2001; Bogorni nd Vendrmim 2003, 2005). However, methods used to otin the extrcts nd evlute the ioctivity in these studies differ considerly from those used in this study, mking it difficult to compre with the dt otined here. Some of these studies refer to queous or orgnic extrcts otined y different methods. the experimentl techniques vry etween tests with corn leves treted dily with the extrcts nd the extrct ddition to the rtificil diet. The concentrtions used, the evlution time nd ge of lrve re lso importnt fctors. Despite ll these vriles, the effects oserved in lrve fter ingestion of food treted Tle 1. Mens of weight nd mortlity (± stndrd error) of Spodopter frugiperd lrve fed on rtificil diet treted with 1% frctions of Trichili pllid nd Trichili pllens leves nd stems, fter ten dys. Pircic, Brzil. Tretment Weight ± SE (mg)¹ Mortlity ± SE (%)² Trichili pllid-dic-stems ± ± 0.38 Trichili pllid-dic-leves ± ± 1.04 Trichili pllid-etac-stems ± ± 1.04 c Trichili pllid-hex-leves ± ± 0.00 c Trichili pllid-etac-leves ± c 1.04 ± 1.04 c Trichili pllid-hex-stems ± c 1.04 ± 1.04 c Control ethnol ± c 1.04 ± 1.04 c Control cetone ± c 1.04 ± 1.04 c Control wter ± c 1.04 ± 1.04 c Trichili pllid-ha-stems ± c 0.00 ± 0.00 c Trichili pllid-ha-leves ± c 0.00 ± 0.00 c F p < < Trichili pllens-dic-stems 4.31 ± ± 2.62 Trichili pllens-etac-stems ± ± 1.99 Control ethnol ± ± 1.04 Control cetone ± ± 0.00 Trichili pllens-ha-stems ± c 1.04 ± 1.04 Trichili pllens-hex-stems ± c 6.25 ± 3.61 Control wter ± c 0.00 ± 0.00 F p < < Trichili pllens-hex-leves ± ± 1.67 Trichili pllens-dic-leves ± ± 4.25 Trichili pllens-etac-leves ± ± 4.86 Trichili pllens-ha-leves ± ± 0.00 Control ethnol ± ± 1.20 Control cetone ± ± 1.67 Control wter ± ± 4.86 F 2.63 p Mens followed y the sme letter, in the column, do not differ (Tukey, p 0.05); 1 GLM, F test (p 0.05); ² GLM, chi-squred test (p 0.05); DIC: dichloromethne; ETAC: ethyl cethte; HEX: hexne; HA: hydrolcoholic.

4 36 Revist Colomin de Entomologí Angelin Mri Mrcomini Giongo et l. Tle 2. Mens of weight nd mortlity (± stndrt error) of Spodopter frugiperd lrve fed on rtificil diet treted with 1% frctions of Toon cilit leves, stems nd fruits, fter ten dys. Pircic, Brzil. Tretment Weight ± SE (mg)¹ Mortlity ± SE (%)² Toon cilit-dic-leves ± ± 0.10 Toon cilit-dic-fruits ± ± 0.10 Toon cilit-etac-stems ± 6.63 c 1.04 ± 0.10 Toon cilit-dic-stems ± cd 0.00 ± 0.00 Toon cilit-ha-leves ± cde 0.00 ± 0.00 Toon cilit-etac-leves ± 9.15 cde 0.00 ± 0.00 Toon cilit-hex-stems ± cde 0.00 ± 0.00 Toon cilit-hex-leves ± 9.53 cde 0.00 ± 0.00 Toon cilit-hex-fruits ± cde 0.00 ± 0.00 Toon cilit-etac-fruits ± cde 0.00 ± 0.00 Toon cilit-ha-fruits ± 5.46 cde 1.04 ± 0.10 Control wter ± cde 0.00 ± 0.00 Toon cilit-ha-stems ± cde 0.00 ± 0.00 Control ethnol ± de 1.04 ± 0.10 Control cetone ± e 2.08 ± 0.14 F 5.98 p < Mens followed y the sme letter, in the column, do not differ (Tukey, p 0.05); 1 GLM, F test (p 0.05); ² GLM, chi-squred test (p 0.05); DIC: dichloromethne; ETAC: ethyl cethte; HEX: hexne; HA: hydrolcoholic. with extrcts of these plnt species re similr: reduction in lrvl weight gin, prolonged lrvl stge, smller pupe, nd concentrtion-dependent mortlity. The To. cilit extrcts, however, minly cused sulethl effects, with low mortlity even t higher concentrtions, s reported y Rodriguez nd Vendmim (1997) for S. frugiperd lrve. The most significnt effect is the low lrvl weight gin. Some frctions cused lrge reductions in lrvl weight few dys fter feeding on treted diets. This my e result of reduced consumption due to phgodeterrent ctivity, or low nutritionl dequcy of the food y the presence of xenoiotics. Another possiility is post-ingestion toxic effect, cusing poor utiliztion of food y insects or inhiiting importnt vitl processes, cusing developmentl dely. However, the mixture of compounds present in the frctions of the plnt extrcts mkes it difficult to conduct more detiled study of the mode of ction. The effect ginst insects of the genus Trichili hs een ttriuted to group of tetrnortriterpenoids tht includes hirtin (Xie et l. 2004) nd the trichilins (Chmpgne et l. 1989; Nktni 2001). The limonoids from To. cilit were relted to the phytochemicl resistnce to the shoot orer Hypsipyl (Agostinho et l. 1994); nd tooncilin, 6-cetoxitooncilin (Krus et l. 1978) nd cedrelone (Arnson et l. 1987; Koul nd Ismn 1992) hve insecticidl nd phgodeterrent properties. The low weight gin oserved in lrve fter ingestion of dichlorometne frctions (except for T. pllens leves) is n indiction tht this solvent is more efficient in the extrction of ctive compounds of these Melicee species. For exmple, Pérez-Flores et l. (2012) used dichloromethne to extrct limonoids from leves of some plnts of this fmily. For Tut solut (Meyrick, 1917) (Lepidopter: Gelechiide), the dichloromethne extrct of T. pllid leves ws lso more efficient thn hexne nd methnol extrcts in hexne (Cunh et l. 2005). Dichloromethne is solvent of medium polrity, extrcting hydrophoic nd mphipthic compounds, thus covering lrge mount of compounds (Ferrcini et l. 2005). This feture is suject to the chemicl composition of ech plnt species; tht is, dichloromethne my not e good solvent when polr compounds re undnt. For Melicee species studied, however, this solvent ws very efficient. Effect on lrvl growth nd survivl. When lrve were ex posed to the tretments during the entire lrvl stge, mong frctions with higher ioctivity on S. frugiperd (dichloromethne frctions of T. pllens stems, T. pllid leves nd stems, nd To. cilit leves nd fruits), only the dichloromethne frction of T. pllens stems cused lrvl mortlity, which ws different from tht recorded in control. The frctions of T. pllid leves nd stems nd To. cilit leves did not differ from the frction of T. pllens stems, ut lso did not differ from the control. The frction of T. pllens stems nd the frction of T. pllid leves significntly ffected the lrvl growth, cusing prolonged lrvl stge in pproximtely six to seven dys compred to the control. In ddition, pupl weight ws lower thn the control with these two tretments nd with the frction of T. pllid stems (Tle 3). As previously verified, the frctions of To. cilit leves nd fruits were those tht lest ffected S. frugiperd survivl nd growth. A chrcteristic of mny Melicee extrcts is slow nd crescent effect over time, justifying the incresed mortlity oserved in this ssy compred to the previous one, which ws only ten dys long, minly of T. pllens stems nd of T. pllid leves nd stems. Sulethl effects were lso oserved, s delyed development, chrcterized y lrvl stge of longer dur tion, nd pupl weight reduction. Bogorni nd Vendrmim (2005) oserved longer durtion of the lrvl stge of S. frugiperd

5 Melicee frctions ginst Spodopter frugiperd 37 Tle 3. Lrvl mortlity, lrvl stge durtion nd pupe weight with 24 hours (± stndrd error) of Spodopter frugiperd fed on rtificil diet treted with 1% dichloromethne frctions of Trichili pllid, Trichili pllens nd Toon cilit. Pircic, Brzil. Tretment Mortlity ± SE (%) 1 Durtion ± SE (dys)² Pupe weight ± SE (mg)³ Trichili pllens-stems ± ± ± Trichili pllid-leves ± ± ± 0.91 Trichili pllid-stems ± ± ± 9.84 Toon cilit-leves ± ± ± c Toon cilit-fruits ± ± ± 4.27 c Control 5.00 ± ± ± 5.03 c F p < < < Mens followed y the sme letter, in the column, do not differ (Tukey, p 0.05); 1 GLM, chi-squred test (p 0.05); 2 GLM, F test (p 0.05), considering survivors lrve, until reching puption; ³ GLM, F test (p 0.05); ll survivor lrve reched puption. fter ingestion of corn leves treted with queous extrcts of T. pllid leves nd stems, nd of queous extrcts of T. pllens leves. Torrecills nd Vendrmim (2001) found the sme effects with queous extrcts of T. pllid stems. Such effects my e due to low food intke, cused y phgodeterrence or nutritionl indequcy of the food sustrte (Rodríguez nd Vendrmim 1998), or y devition of prt of the food ingested to detoxifiction metolism (Tnzuil nd McCffery 1990). With low food intke nd low iomss conversion, the insect tkes longer to rech the criticl weight for ecdysis, which implies n increse of the lrvl stge durtion. Effect on nutritionl physiology. The nlysis of covrince indicted the effect of tretments nd covrites, ut there ws no interction etween the two fctors, for ny of the nutritionl indices. Thus, the dt were sujected to n nlysis of vrince. The reltive consumption rte (RCR), which indictes the mount of food ingested y lrv per grm of ody weight per dy, incresed fter ingestion of the frction of T. pllid stems. The RCR ws reduced in the lrve tht ingested diets contining frction of To. cilit leves, nd the other frctions (T. pllid leves, T. pllens stems nd To. cilit fruits) did not differ from the control (Tle 4). The reltive metolic rte (RMR), which indictes the mount of food spent y the lrv in metolism per grm of ody weight per dy, incresed fter ingestion of diet contining frctions of To. cilit fruits nd T. pllid stems. However, the reltive growth rte (RGR), which indictes the gin of iomss y the insect in reltion to ody weight per dy, ws reduced fter ingestion of ll the frctions, nd the gretest reduction ws cused y the frction of T. pllid leves (Tle 4). This my e due to low food intke or to toxic effect cused y the frctions, resulting in the use of food for purposes other thn growth, such s detoxifiction enzymes synthesis. The efficiency of conversion of ingested food (ECI), which is the percentge of ingested food tht is converted into iomss, ws reduced in lmost ll tretments, except only the frction of To. cilit leves, the only one tht does not differ from the control (Tle 5). The efficiency of conversion of digested food (ECD), which is the estimted per centge of ssimilted food tht ws converted into iomss, decresed fter ingestion of diet contining frctions of To. cilit fruits nd T. pllid leves nd stems (Tle 5). The low conversion of food into iomss justifies the dely in lrvl development oserved with most of the frctions, nd indictes tht they cuse toxic effects, except for the frction of To. cilit leves, which cused no chnge in Tle 4. Nutritionl indices of Spodopter frugiperd fourth instr fed on rtificil diet contining 0.1% (1,000 ppm) dichloromethne frctions of Trichili pllid, Trichili pllens nd Toon cilit, fter four dys. Pircic, Brzil. Tretment RCR (g/g/di) RMR (g/g/di) RGR (g/g/di) Trichili pllid-stems ± ± ± Trichili pllid-leves ± ± c ± c Trichili pllens-stems ± ± cd ± Toon cilit-fruits ± ± ± Control ± ± cd ± Toon cilit-leves ± c ± d ± F p < < < Mens followed y the sme letter, in the column, do not differ (ANAVA, Tukey, p 0.05); RCR = reltive consumption rte (grms of food consumed per grm of ody weight per dy); RMR = reltive metolic rte (grms of food spent in metolism per grm of ody weight per dy); RGR = reltive growth rte (grms of iomss cquired per grm of ody weight per dy).

6 38 Revist Colomin de Entomologí Angelin Mri Mrcomini Giongo et l. Tle 5. Nutritionl indices of Spodopter frugiperd fourth instr fed on rtificil diet contining 0.1% (1,000 ppm) dichloromethne frctions of Trichili pllid, Trichili pllens nd Toon cilit, fter four dys. Pircic, Brzil. Tretment ECI (%) ECD (%) AD (%) MC (%) Trichili pllid-leves ± ± ± ± Trichili pllens-stems ± ± ± ± Trichili pllid-stems ± ± ± ± Toon cilit-fruits ± ± ± ± Control ± ± ± ± Toon cilit-leves ± ± ± ± F p < < < Mens followed y the sme letter, in the column, do not differ (ANAVA, Tukey, p 0.05); ECI = efficiency of conversion of ingested food; ECD = efficiency of conversion of digested food; AD = pproximte digestiility; MC = metolic cost (not nlyzed, the sme of ECD). ECI nd ECD. Frctions tht cused reduction in ECD lso cused incresed metolic cost (MC), i.e., the percentge of food metolized into energy for the mintennce of life, incresed. However, none of the frctions cused chnge in pproximte digestiility (AD), which is the percentge of ingested food effectively ssimilted y the insect (Tle 5). This indictes tht food ws well ssimilted y the lrve, ut ws minly used for metolism since the iomss conversion ws low. In ll the frctions the mount of diet consumed decresed y more thn hlf compred to the control (Fig. 1). As result of food intke reduction, there ws lso reduced feces production nd low weight gin, s well s smller mount of ssimilted nd metolized food. Lrve tht ingested diet contining the dichloromethne frction of To. cilit leves showed the lest mount of food metolized; tht is, the highest mount of food ws used for growth nd not metolism. Despite the reduction of totl food consumption, the reltive consumption to ody weight (Tle 6) ws ltered only with two frctions, incresing fter ingestion of frction of T. pllid stems, nd decresing with the frction of To. cilit leves. In the first cse, the increse indictes tht there is not primry phgodeterrence (chemoreceptormedited), nd the ECI nd ECD reduction indictes tht there is post-ingestion toxic effect, which cn e considered se condry phgodeterrence (reduced consumption cused y toxicity) (Mordue (Luntz) nd Blckwell 1993). For the second frction, phgodeterrence is responsile for reduction in reltive consumption, since there ws no chnge in the efficiency of food conversion, in metolic cost or in reltive metolic rte, excluding the possiility of toxic effects, so the phgodeterrence is possily primry. Therefore, for lrve 0,14 c 0,12 0,10 Grms 0,08 0,06 0,04 0,02 c c c 0,00 Ingested food Feces Assimilted food Weight gin Metolized food Control Toon cilit - Leves Trichili pllens - Stems Trichili pllid - Leves Toon cilit - Fruits Trichili pllid - Stems Figure 1. Mens (± stndrd error) of ingested food (F = , p < ), feces (F = , p < ), ssimilted food (F = 70.01, p < ), weight gin (F = , p < ) nd metolized food (F = 18.42, p < ) y Spodopter frugiperd fourth instr fed on diet contining 0.1% dichloromethne frctions of Trichili pllens, Trichili pllid nd Toon cilit. Ingested food = feces + ssimilted food; ssimilted food = weight gin + metolized food. Brs with the sme letter, in ech prmeter, did not differ (GLM, Tukey test, p 0.05).

7 Melicee frctions ginst Spodopter frugiperd 39 fed on diets contining the frction of To. cilit leves, despite lower food intke, ingested food ws well used, ut not enough for the growth to e equl to tht of the control lrve. For the other frctions, the lrve continued feeding, proly s n ttempt to compenste for the high metolic cost. Extrcts of other Melicee lso cused chnges in the nutritionl physiology of insects. When fourth instr Spodopter litur lrve were fed on diet contining methnolic extrct of rnches of Trichili mericn (Sesse nd Mocino) Pennington, the RGR, RCR, ECI nd ECD ll decrese. It is likely tht this decrese in consumption rte is due to n phgodeterrent effect of the extrct, nd t lest in prt this contriutes to the decrese in growth rte. However, decrese in ECI nd ECD indictes tht T. mericn extrct lso exhiits some post-ingestion toxicity (Wheeler nd Ismn 2001). Methnolic extrcts of Meli zedrch L. leves reduced RGR, RCR, ECD nd ECI of Cnphlocrocis medinlis Guenée (Lepidopter: Pyrlide), while AD incresed (Senthil-Nthn 2006). In these cses, it seems to e mixture of phgodeterrence nd toxic effects, due to simultneous reduction in consumption nd food conversion. In the present work, this ws not oserved. The only frction tht reduced RCR ws To. cilit leves, ut without reduction in ECI nd ECD; thus the decrese in RGR ws due to reduced consumption, not to toxic effect. The other frctions cused toxic effects (reduced ECI nd ECD), ut cused no reduction in consumption, thus showing no phgodeterrence. Conclusions The dichloromethne frction of T. pllens stems ffects growth nd survivl of S. frugiperd lrve; nd frctions of T. pllid leves, of T. pllid stems, of To. cilit leves nd of To. cilit fruits ffect growth. Bioctivity of the dichloromethne frctions of T. pllens stems, of T. pllid leves, of T. pllid stems nd of To. cilit fruits is chrcterized y toxic effects fter ingestion tht cn e referred to s secondry phgodeterrence, wheres frction of To. cilit leves does not cuse toxicity, ut cuse phgodeterrence, most likely primry. Acknowledgements Thnks to the Instituto Ncionl de Ciênci e Tecnologi - Controle Biorrcionl de Insetos Prgs (INCT-CBIP), to Coordenção de Aperfeiçomento de Pessol de Nível Superior (CAPES) nd to the Fundção de Ampro à Pesquis do Estdo de São Pulo (FAPESP), for finncil support. Literture cited ABDELGALEIL, S. A.; OKAMURA, H.; IWAGAWA, T.; SATO, A.; MIYAHARA, I.; DOE, M.; NAKATANI, M Khynolides, rerrnged phrgmlin limonoide ntifeednts from Khy seneglensis. Tetrhedron 57: AGOSTINHO, S. M.; DAS, M. F.; DA SILVA, G. F.; FERNANDES, J. B.; VIEIRA, P. C.; PINHEIRO, A. L.; VILELA, E. F Limonoids from Toon cilit nd specultions on their chemosystemtic nd ecologicl significnce. 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