Altered dietary nutrient intake maintains metabolic homeostasis in parasitized larvae of the insect Manduca sexta L.

Transcription

1 The Journl of Experimentl iology 2, 65 (21) Printed in Gret ritin The Compny of iologists Limited 21 JE ltered dietry nutrient intke mintins metbolic homeostsis in prsitized lrve of the insect Mnduc sext L. S. N. Thompson*, R.. Redk nd L.-W. Wng nlyticl Chemistry Instrumenttion Fcility nd Deprtment of Entomology, University of Cliforni, Riverside, Cliforni 92521, US *uthor for correspondence t Deprtment of Entomology (e-mil: NelsonT@mil.ucr.edu) ccepted 15 ugust 21 Mnduc sext lrve exhibited ltered food selection over 2- or 3-dy feeding period when prsitized by Cotesi congregt, nd offered choice of two chemiclly defined diets, one contining csein without sucrose nd second with sucrose but no csein. While norml lrve consumed the diets in rtio of pproximtely 2:1 protein:crbohydrte (w/w), prsitized insects consumed rtio of pproximtely 1:1. The ltered nutrient rtio consumed by prsitized insects ws principlly due to decrese in consumption of the protein diet, nd ws only prtilly explined by their lower growth. Conditioning lrve for 1 dy to either one of the choice diets hd little effect on subsequent dietry intke over 2-dy feeding period. Conditioned lrve, regrdless of prsitism, initilly fed on the opposite diet immeditely fter conditioning. lthough this suggests tht the ltered nutrient intke displyed by prsitized insects ws not due to ny filure in their cpcity for dietry selection, these results do not definitively demonstrte n ltered nutrient intke trget by prsitized lrve. Rther, prsitism my compromise dietry selection, resulting in rndom feeding. When prsitized lrve were Summry mintined on severl isocloric diets with vrying rtio of csein nd sucrose, those lrve feeding on the diet with rtio of 1:1 of these nutrients supported the lrgest prsite popultion. Previous investigtion of lrve mintined on single rtificil diet estblished tht prsitized insects disply n berrnt induction of gluconeogenesis, so tht hemolymph trehlose is mintined t level equivlent to tht of norml insects. In contrst, the present results demonstrted tht prsitized lrve offered choice of diets, nd feeding t the ltered nutrient rtio bove, mintin hemolymph sugr but hve the sme level of gluconeogenesis s norml lrve given the sme dietry choice. These investigtions suggest tht ltered food selection by prsitized M. sext lrve mintins metbolic homeostsis nd, moreover, my be dptive for C. congregt, potentilly mximizing the number of prsites developing in single host lrv. Key words: nutrition, metbolic homeostsis, dietry intke, trehlose, gluconeogenesis, prsite, Mnduc sext, Cotesi congregt. Introduction Mny insects when presented with vriety of nturl foods will select nd feed on one or combintion tht results in blnced intke of nutrients (Wldbuer nd Friedmn, 1991; Simpson et l., 1995). Lbortory studies of lepidoptern lrve feeding on rtificil diets hve confirmed these observtions nd demonstrted tht lrve conditioned by feeding on diet deficient in one or more nutrients, will lter compenste by selecting n eqully unblnced diet tht is high in those previously deficient nutrients (Wldbuer et l., 19; Simpson et l., 19). This selection behviour is imed t chieving nutrient intke trget nd optiml growth under specific physiologicl nd environmentl conditions (Schiff et l., 199; Simpson nd Rubenheimer, 1996). One my predict tht lrve offered number of diets, ech nutritionlly indequte, will over long developmentl period consume mounts of nutrients s ner s possible to the optiml blnce (Simpson nd Rubenheimer, 1993; Rubenheimer nd Simpson, 1999). Intke of specific nutrients by insects is regulted through feedbck mechnisms (Simpson nd Rubenheimer, 1996). In lepidoptern lrve, regultion of nutrient intke involves the centrl nervous system (Rowell nd Simpson, 1992), s well s the ction of vrious biogenic mines, for exmple, serotonin (Cohen et l., 19). The chemicl composition of the hemolymph, or blood, provides continuous reding of the insect s nutritionl nd metbolic stte (Simpson nd Rubenheimer, 1993b). The insect ft body, the principl orgn for metbolic processing of digestive products following bsorption, is lso responsible for synthesis of the principl hemolymph sugr, trehlose, discchride of glucose, nd for the synthesis nd storge of glycogen nd ft (Keeley, 195). ecuse the ft body lcks mesoderm-derived lining,

2 66 S. N. Thompson, R.. Redk nd L.-W. Wng the hemolymph generlly contins high levels of metbolites tht reflect ft body intermediry metbolism (Mullens, 195). The level of hemolymph trehlose is importnt for regulting crbohydrte intke. Friedmn et l. (1991) observed dietry selection behvior in reltion to trehlose by the lepidoptern insect Helicoverp ze (oddie). They reported tht lrve with low hemolymph trehlose concentrtions choose diet high in crbohydrte rther thn diet high in protein, nd demonstrted tht this choice could be reversed by injection of trehlose to rise hemolymph sugr. We hve conducted investigtions to exmine the nture of dietry selection behviour, nd to determine how hemolymph trehlose level lters, or is ltered by dietry intke, in fifth-instr lrve of Mnduc sext L. (Thompson nd Redk, 2). Commonly clled the tobcco hornworm, M. sext lrve re pests of tobcco, s well s mny ornmentl nd grden plnts (Reinecke et l., 19). Lrve mintined on semi-synthetic rtificil diet (Ymmoto, 1969) supplemented with different mounts of sucrose nd csein displyed vrible hemolymph trehlose levels, depending on the reltive mounts of these nutrients. When subsequently presented with choice of diets contining either sucrose without csein or csein without sucrose, most lrve with low hemolymph trehlose, pproximtely 2 mmol l 1 or less, selected the high crbohydrte diet, while those with high hemolymph trehlose, pproximtely 3 mmol l 1 or more, selected the high protein diet. In the first cse, lrve switched over to the high protein diet, presumbly hving reched threshold trehlose level of between 2 nd 3 mmol l 1. ll lrve, regrdless of the conditioning tretment, ultimtely consumed n verge of pproximtely 2:1 csein:sucrose (w/w) over the entire developmentl stdium. Studies hve lso been conducted recently to exmine the effects of prsitism by Cotesi congregt (Sy) (= pnteles congregtus) on the metbolism of M. sext lrve (Thompson nd Dhlmn, 1999). C. congregt is gregrious hymenoptern prsitoid (Fulton, 19). The dult femle oviposits into the hemocoel or body cvity of host lrve, the eggs htch nd the prsite lrve feed on the nutrient-rich host hemolymph. The host continues to feed nd develop, but t reduced rtes. Fully mture second instr prsite lrve penetrte through the cuticle of the host to the outside. t emergence these prsites moult into third instr lrve tht spin cocoons, ttch to the externl host cuticle nd metmorphose to the pupl stge. Prsitized host lrve displyed n equivlent or higher hemolymph trehlose level thn norml unprsitized individuls. The trehlose concentrtion in prsitized insects is mintined by significnt elevtion of gluconeogenesis (Thompson nd Dhlmn, 199; Thompson, 2), the de novo or net formtion of crbohydrte from mino cid (Mthews nd vn Holde, 199). This metbolic ltertion my reflect redirection of nutritionl resources from host growth to provide suitble nutritionl blnce for the developing prsite lrve. During these experiments, prsitized nd norml lrve were both mintined under identicl nutritionl conditions. Effects of n ltered intke of specific nutrients on metbolism, for exmple the reltive mounts of crbohydrte nd protein, were not considered. It my be, for exmple, tht consumption by prsitized lrve of diet higher in crbohydrte would void the costly energetic expenditure of de novo hemolymph sugr formtion nd in tht mnner be dptive for prsite development. Lower growth nd decresed totl food consumption hve been reported for numerous prsitized lepidoptern lrve (dmo, 199; Poulin, 1995; Quickie, 1997; Thompson nd Hgen, 1999). Studies imed t determining how nutritionl qulity ffects host nd prsite growth nd development, however, re lcking. ecuse nothing is known bout possible chnges in the intke of specific nutrients in response to prsitism, or how such chnges my medite the physiology of the prsitized host, the present study ws conducted to exmine the effects of prsitism on nutrient intke nd crbohydrte metbolism by fifth-instr M. sext, nd to determine the interctions between nutrient intke, metbolism nd growth. Moreover, we exmined the bove interctions in norml nd prsitized M. sext lrve conditioned t the beginning of the fifth instr on diets of different nutrient composition, since previous studies by others demonstrted tht conditioning or prior dietry experience within stdium my ffect subsequent nutrient intke by lepidoptern lrve (Simpson et l., 19; Friedmn et l., 1991). Mterils nd methods Insect culture nd composition of diets Stock colonies of Mnduc sext L. were rered t 2 C under 16 h: h light:drk photocycle on n rtificil diet principlly contining Torul yest nd whet germ (ell nd Jochim, 1976). During the experiments, insects were rered through the fourth stdium on the bove rering diet, nd upon moulting to the fifth stdium were trnsferred onto chemiclly defined synthetic diet (hmd et l., 199), modified to contin csein nd sucrose s the principl nutrient sources of protein nd crbohydrte, respectively. These nutrients were included t 12 g l 1. In ddition, the diet contined: Wesson s slts, 15 g l 1 ; cholesterol, g l 1 ; scorbic cid, 6.7 g l 1 ; cysteine HCl, 1 g l 1 ; vitmin mixture, 215 mg l 1 ; inositol, 2 mg l 1 ; choline chloride, 9 mg l 1 ; β crotene, 2 mg l 1 nd linseed oil, 3.7 ml l 1. Sorbic cid, 2gl 1 ; methyl-p-hydroxybenzote, 2 g l 1 nd 32 ml l 1 of 1 % formlin were included s ntimicrobil gents. The nutrients were principlly obtined from Nutritionl iochemicls (Clevelnd, OH, US) nd ioserve (Frenchtown, NJ, US). Stock colonies of the prsite, Cotesi congregt Sy, were rered on host lrve prsitized in the second stdium. dult prsites were mintined in 3 l glss jrs closed over the top with muslin fbric. dults were fed honey spred on smll cotton blls plced on the bottom of the jr, nd were provided with wter in smll vils with cotton roll wicks. For the nutritionl investigtions, phrte, developmentlly synchronous fourth-instr host lrve (eckge et l., 199)

3 Dietry intke nd metbolic homeostsis in M. sext 67 were plced individully in jr of prsites nd crefully observed. Lrve were prsitized 2 times, to ensure tht sufficient eggs were deposited in ech host for mximum prsite lod, i.e. the mximum number of emerged prsites, for fourth-instr M sext, s reported by others (lleyne nd eckge, 1997). It ws not possible to regulte, or to know precisely the number of times tht individul hosts were prsitized. It my be, therefore, tht some individuls within single tretment group received more prsite eggs thn others in the sme group. However overll, within ech experiment, insects for ll tretments were prsitized in the sme fshion nd t the sme time, suggesting the likelihood tht the distribution of prsite eggs ws similr mong individuls within ll groups. Moreover, fter being prsitized, host lrve subsequently used for the dietry selection experiments were returned to the norml whetgerm rering diet nd, upon moulting to the fifth stdium, were rndomly selected nd plced on the experimentl diets. Following the feeding experiments, prsitized lrve were dissected to observe the presence of developing prsites nd confirm tht successful prsitism hd occurred. Dietry selection Unconditioned selection Newly moulted fifth-instr M. sext lrve were individully offered smll blocks, pproximtely cm 3, of the experimentl choice diets, i.e. the protein diet nd the sucrose diet, in disposble plstic Petri plte (1 cm dimeter). The two diet blocks were rrnged pproximtely 5 cm prt, ech n equl distnce from the center of the plte. Individul lrve were then plced between the two blocks, in the center of the plte, nd ligned with the blocks to prevent ny bis towrd either block. 2 lrve were fed for 2 dys, then weighed nd the mount of ech diet consumed determined. ecuse subsequent experiments on the effects of conditioning lrve on selection involved totl feeding period of 3 dys, second non-conditioning experiment ws conducted with lrve feeding for 3 dys. Vlues were recorded s wet mss in both experiments. Dehydrtion of the choice diets mounted to less thn 1 % over the feeding period. This wter loss ws monitored in Petri pltes contining the diets but without lrve, nd ws ccounted for t the end of the experiments in determining the diet consumed by the lrve (Simpson et l., 19). We ssumed tht the feeding behviour of newly moulted fifth-instr lrve ws unffected by prior dietry experience in erlier stdi. Other studies, however, hve indicted tht this my not be the cse, nd tht only newly htched first instr lrve re truly nïve (Stdler nd Hnson, 197). Newly moulted fifth-instr M. sext lrve rered on plnt mteril subsequently demonstrted strong preferences for the plnt or extrcts of the plnt on which they hd been mintined erlier, nd this ws recently confirmed (Cmpo et l., 21). Stdler nd Hnson (197) lso reported tht lrve rered on whetgerm-bsed rtificil rering diet lcking plnt mteril displyed no preference for the rtificil diet or for ny other food source tested fter moulting to the fifth stdium. lthough recent studies with fifth-instr M. sext demonstrte tht nutritionl experience in erlier stdi ffects consumption nd growth following moulting regrdless of dietry composition (Wood, 1999), ll lrve used in the present studies were rered on the sme diet until the fifth stdium. In our view, therefore, the ssumption of nutritionl nivety for newly moulted fifthinstr lrve used in the conditioning experiments ws resonble. Conditioned selection Newly moulted fifth-instr lrve were trnsferred from the rering diet to one of the two choice diets, contining either csein or sucrose, nd were conditioned by feeding on the diet for 1 dy in Petri plte. Following the conditioning period, the lrve were individully given the choice of the two diets presented together s described bove, nd were llowed to feed for n dditionl 2 dys. Duplicte experiments, ech consisting of 2 or 25 lrve, were crried out nd dt were collected s described bove. Conditioned short-term selection The short-term effect of conditioning on subsequent feeding ws exmined in similr mnner to tht described (Friedmn et l., 1991). Groups of 2 newly moulted fifth-instr lrve were plced on either the sucrose diet or the csein diet for 1 dy s bove. Following this conditioning period, lrve were trnsferred to Petri pltes contining smll block of ech diet nd were llowed to self-select for h. The proportion of lrve feeding on ech diet ws recorded t 1 min intervls for the first 1.5 h, nd then every 2 min for n dditionl 2.5 h. t ny one time, 2 5 lrve were not feeding on either diet nd these lrve were not included in the clcultions. Dt were plotted s the percentge of lrve feeding on ech diet versus time, with the two reltionships, crbohydrte nd protein, inversely relted. ll dietry selection experiments were conducted t room temperture, pproximtely 21 C. Host nutritionl sttus nd prsite burden To exmine the effect of the rtio of dietry protein to crbohydrte on prsite growth nd development, groups of seven M. sext lrve were prsitized s described bove nd immeditely trnsferred onto diets with the following rtios (w/w) of csein to sucrose:.25:1.75,.5:1.5, 1.:1., 1.5:.5 nd 2.:. The rtios were bsed on the mount of csein nd sucrose in the stock chemiclly defined diet, which contined ech nutrient t level of 9 g l 1. ll diets were isocloric nd contined the sme totl mount of nutrient. During the fifth stdium, s prsite emergence ensued for insects on ech diet, the experiment ws terminted nd the host lrve were dissected. The prsite burden, the totl number nd mss of prsites emerged s well s mture second-instr prsites tht hd not yet emerged, ws determined. The mount of diet consumed by ech host lrve

4 6 S. N. Thompson, R.. Redk nd L.-W. Wng ws lso determined. Prsite mss nd the mount of csein nd sucrose in ech diet consumed were expressed s dry mss. Estimtion of gluconeogenesis nd hemolymph trehlose level The net gluconeogenic flux of norml nd prsitized M. sext lrve, llowed to self-select between the csein nd sucrose diets s described bove, ws determined by nucler mgnetic resonnce spectroscopic (NMR) nlysis of pyruvte cycling nd the 13 C-enrichment of lnine nd trehlose following dministrtion of [2-13 C]pyruvte, s recently described (Thompson, 2b). riefly, lnine ws 13 C-enriched t C2 nd C3 by trnsmintion of [2,3-13 C]pyruvte formed following crboxyltion of the dministered isotopiclly substituted substrte to oxlocette, rndomiztion of the 13 C-enrichment t the fumrse-ctlyzed step of the tricrboxylic cid cycle (TC) cycle nd formtion of [2,3-13 C]phosphoenolpyruvte. The lnine C3/C2 13 C-enrichment rtio is mesure of the extent of pyruvte cycling. If rndomiztion of 13 C within the TC cycle is similr between tretment groups then their lnine rtios re directly comprble. The degree of 13 C- rndomiztion is indicted from the enrichment of glutmte, glutmine nd the glutmyl moiety of glutthione (Gl x ), which re sequentil byproducts of TC cycle metbolism following rndomiztion. Subsequent to the formtion of [2,3-13 C]phosphoenolpyruvte, glucose nd trehlose re synthesized vi the gluconeogenic pthwy. Phosphoenolpyruvte C3 gives rise to trehlose C1 nd C6, while phosphoenolpyruvte C2 gives C2 nd C5 of trehlose. The 13 C-enrichment of trehlose C1 nd C6 reltive to tht of lnine C3 is mesure of the gluconeogenic flux reltive to the glycolytic flux, s both the trehlose nd the lnine 13 C-enrichments re derived in the sme fshion (Thompson, 2b). This metbolism is summrized in Fig.. Due to pentose cycling following gluconeogenesis, however, the 13 C-enrichment of trehlose C1 is reduced reltive to tht of C6 (Thompson, 1999), nd the 13 C rtio [2trehlose C6/lnine C3] ws employed s the mesure of gluconeogenic flux, trehlose C6 being unffected by pentose cycling. Hemolymph trehlose concentrtion ws estimted by compring the NMR signl intensities of the individul crbons of trehlose with those of n internl stndrd, ssuming nturl 13 C bundnce of 1.1 % for trehlose C. The bundnce of 13 C in trehlose C2 nd C5 ws estimted in reltion to trehlose C1 nd C6 using the Gl x C2/C3 13 C-enrichment rtio tht reflects the non-symmetric 13 C- distribution in trehlose. In this cse, ny contribution of cytoplsmic crboxyltion to trehlose formtion ws not considered. Previous investigtion indicted significnt, but very smll, cytoplsmic contribution (Thompson nd Redk, 2). dministrtion of [2-13 C]pyruvte, preprtion of hemolymph nd NMR nlysis [2-13 C]pyruvte (=99 tom %), obtined from Cmbridge Isotope Lbortories (Woburn, M, US), ws dministered to norml nd prsitized lrve by injection (5 µmol g 1 fresh mss) of n queous solution (1 mg 5 µl 1 ) into the dorsl vessel. Hemolymph ws collected from incisions in the prolegs 3.5 h post-injection t stedy stte, s previously outlined (Thompson nd Redk, 2). Hemolymph smples were deproteinized with perchloric cid, neutrlized nd nlyzed by NMR (Thompson, 2; Thompson, 2b). Duplicte experiments were conducted. NMR nlyses were conducted in GE QE 3 spectrometer t 75. MHz s outlined previously (Thompson, 2,b)). Quntittion of signl intensities ws conducted by the method of Christensen et l. (197). 13 C-enrichment rtios were clculted s described bove. Sttisticl nlyses Unconditioned dietry selection To determine the effect of prsitism on growth nd consumption of M. sext lrve, one-wy nlysis of covrince (NCOV) ws utilized with initil lrvl mss s the covrite, nd prsitism s the min tretment effect (prsitized versus norml) (Tble 1). Throughout the sttisticl nlyses the initil mss employed ws the mss of lrve t the beginning of the fifth stdium nd fter prsitiztion. Prsitized nd norml lrve hve similr msses t this time. Dependent vribles estimting growth (finl mss including the prsite biomss) nd consumption (consumption of protein nd crbohydrte diets) were nlyzed seprtely. n NCOV ws employed to correct for ny potentil size effect on the dependent vribles. Tretments were pplied to replicte group of 2 insects. Conditioned dietry selection To determine the effects of prsitism with dietry conditioning on growth nd consumption of M. sext lrve, two-wy NCOV ws utilized with initil mss s the covrite. Prsitism (prsitized versus norml) nd type of conditioning diet (crbohydrte versus protein) were considered s min-effect tretments. Dependent vribles estimting growth nd consumption were nlyzed seprtely, with ech tretment pplied to replicte group of 2 insects. The results from the conditioning experiment indicted tht diet consumption, nd growth (finl mss) were ffected by prsitism (Tble 2). s with other nutritionl studies (lu et l., 197; Horton nd Redk, 1993), our dt showed tht the effect of prsitism on consumption ws difficult to evlute due to the known effect of size on consumption, with lrger insects consuming more food. Consequently, to distinguish the effects of prsitism nd conditioning from size-medited tretment effects on consumption of protein diet nd crbohydrte diet, seprte two-wy NCOVs were utilized with finl lrvl mss s the covrite, nd prsitism nd dietry conditioning s the min tretment effects. To provide for the most generl model, the dt from the duplicte experiments were combined.

5 Dietry intke nd metbolic homeostsis in M. sext 69 Tble 1. NCOV summry demonstrting the effects of prsitism on diet consumption nd growth of the host Mnduc sext given dietry choice (see Fig. 1) d.f. Men squre F P Experiment 1 Protein diet consumption Prsitism <.1 Initil mss (covrite) =.336 Error Crbohydrte diet consumption Prsitism =.66 Initil mss (covrite) =.171 Error Finl mss Prsitism <.1 Initil mss (covrite) =.35 Error Experiment 2 Protein diet consumption Prsitism <.1 Initil mss (covrite) =.27 Error Crbohydrte diet consumption Prsitism =.5 Initil mss (covrite) =.75 Error Finl mss Prsitism =.337 Initil mss (covrite) =.12 Error Host nutritionl sttus nd prsite burden The dt for the totl number of prsites in hosts mintined on the vrious diets were exmined by the Kruskll Wllis nonprmetric nlysis of vrince (NOV) with the rtio of dietry csein to sucrose used s min-effect tretment t five levels (csein:sucrose,.25:1.75,.5:1.5, 1.:1., 1.5:.5 nd 2.:). Tretments were replicted 5 7 times. fter estblishing significnt difference mong rtios, the Nemenyi multiple-rnge test ws pplied to determine significnt differences between mens. In ddition, threedimensionl surfce plots were generted to visulize the reltionship between dietry levels of csein nd sucrose with the prsite burden. These plots were creted using SS (version, 1999, SS Institute Inc. Cry, NC, US). The dt were used to crete mtrix of 9 points by interpolting simple liner function describing the reltionship between dietry nutrient levels onto rectngulr grid estimting prsite burden (PROC G3GRID). The resulting 9 point dt mtrix ws subsequently plotted in three dimensions (PROC G3D) or s contoured surfce (PROC GCONTOUR). In the cse of prsite mss, the dt were nlyzed by onewy NOV fter determining tht there ws no reltionship between prsite mss nd initil host mss. Following Tble 2. NCOV summry demonstrting the effects of dietry conditioning nd prsitism on diet consumption nd growth of the host Mnduc sext given dietry choice (see Fig. 2) d.f. Men squre F P Experiment 1 Protein diet consumption Conditioning =.617 Prsitism <.1 Interction conditioning/ =.925 prsitism Initil mss (covrite) =.2537 Error Crbohydrte diet consumption Conditioning =.673 Prsitism =.13 Interction conditioning/ =.3135 prsitism Initil mss (covrite) =.529 Error Finl mss Conditioning =.332 Prsitism <.1 Interction conditioning/ =.399 prsitism Initil mss (covrite) =.1727 Error Experiment 2 Protein diet consumption Conditioning 1.17 =.919 Prsitism <.1 Interction conditioning/ =.2117 prsitism Initil mss (covrite) =.3 Error Crbohydrte diet consumption =.2 Conditioning =.253 Prsitism =.67 Interction conditioning/ =.63 prsitism Initil mss (covrite) Error 95 Finl mss Conditioning <.1 Prsitism <.1 Interction conditioning/ =.515 prsitism Initil mss (covrite) <.1 Error significnt tretment effect, dt were subjected to the Ryn Einot Gbriel Welch multiple-rnge test. Pyruvte cycling nd gluconeogenesis To evlute the effects of prsitism, one-wy NOV ws conducted on the dt for estimted percentge 13 C content in

6 7 S. N. Thompson, R.. Redk nd L.-W. Wng Fig. 1. Effects of prsitism by Cotesi congregt on diet consumption nd growth of the host Mnduc sext given dietry choice. Nutrient consumption is shown s bivrite plot of protein nd crbohydrte consumption. Vlues re mens ± lestsqures S.E.M.; horizontl brs refer to protein diet nd verticl brs to crbohydrte diet. Vlues followed by different letters re sttisticlly different for protein diet or crbohydrte diet, indicted by the directionl error br. Tringles nd lower cse letters refer to Experiment 1, where lrve were fed for 2 dys. Circles nd upper cse letters refer to Experiment 2, where lrve were fed for 3 dys. Growth for the two Crbohydrte diet consumption (g) 6 2 Experiment 1: Norml Prsitized Protein diet consumption (g) experiments is shown in the ccompnying br grph. Newly moulted norml (filled brs) nd prsitized (open brs) fifth-instr lrve were given choice of two synthetic rtificil diets, one contining sucrose without csein, nd the other, csein without sucrose, both nutrients t 12gl 1. ll vlues re g wet mss. Dt were nlyzed by NCOV with initil mss s the covrite. For sttisticl summry, see Tble 1. Experiment 2: Norml Prsitized b Finl mss (g) 6 2 Norml Prsitized b Exp. 1 Exp. 2 trehlose. Dt for the 13 C-enrichment rtios, Gl x C2/C3, lnine C3/C2 nd [2trehlose C6/lnine C3], s well s the hemolymph trehlose level, were nlyzed by one-wy NCOV, using initil mss s the covrite; prsitism ws considered to be the min-effect tretment. Fulfilment of sttisticl ssumptions Dt from the feeding experiments tht were nlyzed by NCOV or NOV met the ssumptions of normlity nd homogeneity of vrince bsed on the Shpiro Wilk W test nd ssessment with norml probbility plot. Dt for prsite biomss s well s the biochemicl dt were lso normlly distributed. Dt for prsite burden, however, were not norml, nd non-prmetric nlyses were conducted s described bove. Results Dietry selection Unconditioned selection fter newly moulted fifth-instr M. sext lrve were presented with choice of csein or sucrose diets immeditely upon moulting to the fifth stdium, norml lrve consumed significntly more of the protein diet thn did prsitized lrve over both the 2-dy nd 3-dy feeding periods (Fig. 1, Tble 1). Consumption of the sucrose diet during the 3-dy experiment ws significntly different between norml nd prsitized insects, with prsitized insects consuming less. The difference in crbohydrte consumption, however, ws much smller thn ws observed with protein consumption in both experiments. The men rtios of protein to crbohydrte diets consumed in the h experiment were 3.33±.5 (men ± S.E.M.) for norml nd 1.6±.5 for prsitized lrve. During the 3-dy experiment the rtios were 1.6±.13 (men ± S.E.M.) nd 1.1±.13 for norml nd prsitized nimls, respectively. In both experiments, the difference between the rtios of norml nd prsitized insects ws sttisticlly significnt (Experiment 1, F 1,37 =.53, P=.; Experiment 2, F 1,37 =7.91, P=.7). The finl msses of norml insects were lso significntly greter thn those of prsitized insects (Fig. 1, Tble 1). Conditioned selection Conditioning newly moulted fifth-instr M. sext lrve on either the csein diet or the sucrose diet hd little effect on subsequent dietry choice, but differences were evident between the duplicte experiments (Fig. 2, Tble 2). In Experiment 2, conditioning on the protein diet resulted in proportiontely smll but significnt increse in crbohydrte consumption tht ws not evident in the first experiment. Similrly, in Experiment 2, there ws significnt increse in the finl mss of lrve conditioned on the protein diet. In both experiments, norml lrve consumed significntly more protein diet, pproximtely twofold more, thn did prsitized insects. In the first experiment, the men rtios of protein to crbohydrte diet consumed by norml nd prsitized insects were 1.1±. (men ± S.E.M.) nd 1.19±., respectively (F 1,95 =25.6, P<.1), nd in the second experiment, 1.2±.11 (men ± S.E.M.) for norml lrve nd 1.13±.11 for prsitized insects (F 1,75 =19.37, P<.1). The men finl mss of the prsitized lrve ws significntly less thn tht of norml insects following conditioning on the csein or sucrose diets (Fig. 2, Tble 2). When the bove dt for the conditioning experiments were evluted to determine the effects of conditioning nd prsitism, independently of the reltionship between consumption nd finl mss or size (Tble 3), conditioning ws found to hve significnt effect on subsequent crbohydrte diet consumption

7 Dietry intke nd metbolic homeostsis in M. sext 71 Conditioning Fig. 2. Effects of dietry conditioning () nd prsitism () by Cotesi congregt on diet consumption nd growth of the host Mnduc sext, when given dietry choice. Nutrient consumption is shown s bivrite plot of protein diet nd crbohydrte diet consumption. Vlues re indicted by men ± lest-squres S.E.M.; horizontl brs refer to protein diet nd verticl brs to crbohydrte diet. Vlues followed by different letters re sttisticlly different for the protein diet or crbohydrte diet, indicted by the directionl error br. Tringles nd lower cse letters, nd circles nd upper cse letters, refer to the duplicte Experiments (Exp.) 1 nd 2, respectively. Newly moulted norml nd prsitized fifth-instr lrve were conditioned for 1 dy on synthetic rtificil diet contining sucrose without csein, or csein without sucrose (both nutrients t 12 g l 1 ), nd subsequently given choice of the two diets for n dditionl 2 dys. The effects of conditioning nd prsitism on growth re shown in the insets. ll vlues re g wet mss. Dt were nlyzed by two-wy NCOV with initil mss s the covrite. No significnt interction between conditioning nd prsitism ws evident. For sttisticl summry, see Tble 2. Crbohydrte diet consumption (g) Experiment 1: Norml Prsitized Prsitism Experiment 1: Norml Prsitized b Finl mss (g) Protein diet consumption (g) b Experiment 2: Norml Prsitized b Finl mss (g) Experiment 2: Norml Prsitized Norml Prsitized b Exp. 1 Exp. 2 Norml Prsitized b Exp. 1 Exp. 2 Tble 3. NCOV summry demonstrting the size-medited effects of dietry conditioning nd prsitism on diet consumption by the host Mnduc sext given dietry choice (see Fig. 3) Dependent vrible Effect d.f. Men squre F P Protein diet consumption Conditioning Prsitism Interction-conditioning/prsitism Finl mss (covrite) <.1 Interction-finl mss/conditioning Interction-finl mss/prsitism Interction-finl mss/prsitism/conditioning Crbohydrte diet consumption Conditioning Prsitism Interction-conditioning/prsitism Finl mss (covrite) <.1 Interction-finl mss/conditioning Interction-finl mss/prsitism Interction-finl mss/prsitism/conditioning

8 72 S. N. Thompson, R.. Redk nd L.-W. Wng Crbohydrte diet consumption (g) Conditioned on protein diet Conditioned on crbohydrte diet C Control Protein diet consumption (g) Conditioned on crbohydrte diet D Conditioned on protein diet Control 6 Prsitized 2 Prsitized Finl mss (g) Finl mss (g) Fig. 3. Size-medited effects of dietry conditioning nd prsitism by Cotesi congregt on consumption of protein diet nd crbohydrte diet by the host Mnduc sext given dietry choice. Results for individul lrve re shown with regression lines depicting the reltionships between size nd consumption for ech tretment level. () Effects of conditioning on crbohydrte consumption. For lrve conditioned on the protein diet (tringles), consumption=.291 finl mss; for lrve conditioned on the crbohydrte diet (circles), consumption=.62 finl mss. () Effects of conditioning on protein consumption. For lrve conditioned on the protein diet (tringles), consumption=1.372 finl mss; for lrve conditioned on the crbohydrte diet (circles), consumption=.961 finl mss. (C) Effects of prsitism on crbohydrte consumption. For prsitized nimls (tringles), consumption=.291 finl mss; for norml nimls (circles), consumption=.95 finl mss. (D) Effects of prsitism on protein consumption. For prsitized nimls (tringles), consumption=1.372 finl mss; for norml nimls (circles), consumption=2.319 (finl mss). The results of the NCOVs re shown in Tble 3. (Fig. 3), but not on protein diet consumption (Fig. 3). In the former cse, the slopes of the regression lines for the reltionships between conditioning nd consumption were significntly different, while in the ltter cse, the slopes were not different. oth crbohydrte nd protein diet consumption were significntly ffected by insect size (significnt covrite, Tble 3). Lrger insects consumed more of both diets (Fig. 3,), but the effect of insect size on consumption of the crbohydrte diet ws medited by conditioning ( significnt tretment by covrite interction ws detected, Tble 3). The effect of conditioning ws stronger for smller lrve (pproximtely 5 g or less), thn for lrger insects (pproximtely 7.5 g or more). In the cse of smller lrve, consumption of the crbohydrte diet ws significntly greter for lrve conditioned on the protein diet, thn for insects tht were conditioned on the crbohydrte diet (Fig. 3). There ws significnt interction between conditioning nd finl mss (Tble 3). s lrvl size incresed, the difference in crbohydrte diet consumption between conditioning tretments becme negligible. The effect of lrvl mss on crbohydrte nd protein diet consumption ws medited by prsitism (Tble 3). The increse in consumption of crbohydrte diet (Fig. 3C) nd protein diet (Fig. 3D) with incresed lrvl size ws not s gret for prsitized lrve s ws the cse for norml insects. The slopes of the regression lines for the reltionships between crbohydrte nd protein diet consumption with finl mss of prsitized nd norml lrve were significntly different. In this study, the effects of prsitism on consumption of the crbohydrte diet were most pronounced for individuls t the extremes of the size distribution. With smller lrve, prsitism resulted in higher crbohydrte diet consumption thn ws observed by norml insects of similr mss, while for

9 1 Dietry intke nd metbolic homeostsis in M. sext Fig.. Short-term dietry selection by the host Mnduc sext. Newly moulted fifth-instr lrve were conditioned for 1 dy on synthetic rtificil diet contining sucrose without csein, or csein without sucrose (both nutrients t 12 g l 1 ), nd subsequently given choice of the two diets. (,) The percentge of norml lrve () nd prsitized lrve () choosing the protein (squres) nd crbohydrte (tringles) diets following conditioning on the protein diet. (C,D) The percentge of norml (C) nd prsitized (D) lrve choosing the protein nd crbohydrte diets following conditioning on the crbohydrte diet. Ech grph shows the distribution of 2 lrve. Feeding lrve (%) Time (min) C Protein diet Crbohydrte diet Time (min) D lrger lrve, prsitism resulted in lower crbohydrte diet consumption (Fig. 3C). The effect of prsitism on consumption of protein diet ws most pronounced for lrger lrve nd ws miniml for smller insects (Fig. 3D). Lrger prsitized insects consumed significntly less protein diet thn norml lrve. For both crbohydrte nd protein diet consumption there were no min-effect interctions between prsitism nd conditioning (Tble 3). Short-term selection Lrve conditioned for 1 dy on the csein diet fed predominntly on the sucrose diet when given choice of the csein nd the sucrose diets (Fig.,). The results with prsitized nd norml lrve were comprble, lthough norml lrve begn to switch to the protein diet fter pproximtely 2.5 h. Similrly, both norml nd prsitized lrve conditioned on the sucrose diet selected the csein diet when given the dietry choice (Fig. C,D). Host nutritionl sttus nd prsite burden There were significnt differences in the numbers of prsites developing on hosts mintined on isocloric diets with vrying rtios of dietry csein nd sucrose (χ 2 =22.2, d.f.=, P=.2) (Fig. 5). The number of prsites incresed with incresing protein nd reched mximum when the protein to crbohydrte rtio ws 1:1 (w/w), nd therefter decresed. further Kruskl Wllis nlysis confirmed tht the 1:1 diet supported the highest prsite burden, expressed s numbers of prsites g 1 host mss, fter djusting for finl host mss (χ 2 =19.99, d.f.=, P=.5). There were no significnt differences mong the msses of individul prsites from host lrve rered on ny of the diets, except for the.25:1.75 diet with the lowest level of csein (F,25 =25.5, P<.1, Fig. 5). The totl prsite mss, however, incresed with incresing protein nd ws mximl t the 1:1 protein/crbohydrte rtio (F,25 =17.56, P<.1, Fig. 5). t higher reltive protein levels the totl mss decresed. The results for the effects of dietry nutrient rtio on the totl mss of prsites, therefore, generlly reflected the totl prsite numbers. The three-dimensionl view showing the reltionship between consumption of sucrose nd csein diets with prsite burden suggests complex reltionship, but clerly demonstrtes tht increses in both dietry csein nd sucrose increse prsite burden (Fig. 6). The effect of protein consumption, however, ws significntly greter thn tht of crbohydrte consumption. The lrge pek in the upper left qudrnt ws due to the exceptionlly high number of prsites, 67, in n individul host lrv on the 1.5:.5 diet. Without this contribution the reltionship would hve shown continuous upwrd slope. The men number of prsites in hosts on the 1.5:.5 diet ws 39±77 (men ± S.E.M.), which includes the high vlue. In contrst, the number of prsites in host lrve on the 1:1 diet, the diet supporting the gretest

10 7 S. N. Thompson, R.. Redk nd L.-W. Wng Number of prsites Totl prsite dry mss (g) b b b c bc b.25:1.75.5:1.5 1.:1. 1.5:.5 2.: Dietry csein: sucrose rtio Individul prsite dry mss (mg) Dietry csein (g) Number of prsites Dietry csein (g) Dietry sucrose (g) Fig. 5. Effect of dietry protein/crbohydrte rtio on the numbers nd mss of Cotesi congregt developing in the host Mnduc sext. () Effect of dietry nutrient rtio on totl prsite number. Vlues re mens ± S.E.M. Vlues followed by different letters re sttisticlly different t α=.5 s determined by the Nemenyi multiple-rnge test. () Effect of dietry nutrient rtio on totl (squres) nd individul (tringles) prsite biomss. Vlues re mens ± S.E.M. Vlues followed by different letters re sttisticlly different t α=.5 from those in the sme dt set, s determined by the Ryn Einot Gbriel Welsch multiple-rnge test. ll diets were isocloric nd contined n equivlent totl mount of csein nd sucrose. prsite burden, ws 5±32. Fig. 6, two-dimensionl topogrphicl representtion of these sme dt, shows the predicted reltionship between dietry csein nd sucrose consumption nd prsite burden. The reltionship for the 1:1 dietry rtio nd prsite burden is indicted on the figure, with the ctul experimentl dt for tht rtio shown. The results suggest tht the dietry rtio supporting the gretest number of prsites my be slightly more thn 1:1. Gluconeogenic formtion of hemolymph trehlose ll norml nd prsitized M. sext lrve were glucogenic nd displyed net trehlose formtion vi gluconeogenesis (Tbles 6). The selective nd pired enrichment of trehlose C1 nd C6, nd C2 nd C5, whih re typicl of glucogenesis from [2-13 C]pyruvte (Thompson, 2,b), were clerly evident in the 13 C NMR spectr of hemolymph extrcts (Fig. 7). The degree of rndomiztion of 13 C by the TC cycle ws indicted by the 13 C-enrichment in C2 nd C3 of Gl x Dietry sucrose (g) Fig. 6. The reltionship between dietry protein nd crbohydrte consumption nd the numbers of Cotesi congregt developing in the host Mnduc sext. () Three-dimensionl representtion. () Topogrphicl representtion of. The digonl line shows the predicted reltionship for the 1:1 diet contining n equivlent mount of csein nd sucrose, bsed on ctul experimentl vlues s shown. produced s byproducts of TC cycle metbolism (Tble 5, Fig. ). The Gl x C2/C3 13 C-enrichment rtio ws not significntly different in either experiment, confirming tht the lnine C3/C2 rtio in n ccurte mesure of pyruvte cycling. The mesurements of pyruvte cycling, lnine C3/C2 nd glucogenic flux, [2trehlose C6/ lnine C3], were lso not significntly different between norml nd prsitized lrve. 13 C-enriched metbolites were mixtures of singly enriched isotopomers. No mutiply enriched species were detected in ny of the experiments. Generlly, prsitized nd norml lrve hd similr rnges of hemolymph trehlose concentrtion, despite the difference in the rtio of protein/crbohydrte diets tht the lrve consumed (Tble 5). In experiment 2, prsitized lrve displyed significntly higher men hemolymph sugr level, but this, in prt, reflected the contribution of single insect with n unusully high trehlose concentrtion

11 Dietry intke nd metbolic homeostsis in M. sext 75 Tble. Estimted 13 C in the individul crbons of trehlose in hemolymph extrcts of norml nd prsitized Mnduc sext lrve, given dietry choice 13 C in trehlose (%) Stte of host C1 C2 C3 C C5 C6 Experiment 1 Norml 3.1± ± ± ±1.77.2±.77 Prsitized 2.57±.75.91± ± ± ±.6 F=.32, P=.592 F=.91, P=.372 F=.1, P=.9192 F=2.26, P=.1767 F=1.32, P=.26 Experiment 2 Norml 1.5±.19 2.±.3.9± ±.3 2.7±. Prsitized 1.35± ±.3.91± ±.3 1.3±.21 F=3.36, P=.13 F=1.96, P=.199 F=.1, P=.7551 F=.76, P=.67 F=9.26, P=.16 Vlues re mens ± S.E.M. nd were estimted from the integrted intensities of the individul crbons for trehlose in the 13 C NMR spectrum ssuming nturl bundnce of 1.1% for C. Tble 5. Effects of prsitism on the rtio of protein nd crbohydrte diets consumed nd glucogenic formtion of trehlose nd hemolymph sugr level by the host Mnduc sext given dietry choice Consumption rtio Protein diet/ 13 C-enrichment rtio [Trehlose] Stte of host crbohydrte diet G1 x C2/C3 lnine C3/C2 2trehlose C6/lnine C3 (mmol l 1 ) Experiment 1 Norml 2.3±.19* 1.5±.23.29±. 5.67± ±7.9 Prsitized 1.32±.21* 1.35±.26.22±. 6.51±1. 3.±. Experiment 2 Norml 2.21±.32*.63±.13.32±. 2.36± ±5.11* Prsitized.6±.36*.66±.13.22±..56± ±5.11* Newly molted fifth-instr lrve were given choice of two synthetic rtificil diets, one contining sucrose without csein, nd the other, csein without sucrose. oth nutrients were t 12 g l 1. Vlues re lest-squres mens ± S.E.M. For experiment 1, N=5 (norml), N= (prsitized); for experiment 2, N=5 (norml nd prsitized). *Vlues re significntly different between norml nd prsitized groups within ech experiment. See Tble 6 for sttisticl nlysis. of pproximtely 71 mmol l 1. Hemolymph sugr level in the present experiments ws much more vrible thn ws observed during previous studies where insects were provided with single diet, rther thn being offered choice of diets. Discussion The present study demonstrted dietry selection by fifthinstr M. sext lrve when insects were given choice between two nutritionlly indequte diets, one contining csein without crbohydrte, nd the other sucrose without protein. Norml lrve consistently consumed more of the csein diet reltive to the sucrose diet, which in most cses ment pproximtely twofold more protein. This result is in greement with the dietry rtios of protein nd crbohydrte consumed by mny lepidopern insects feeding on nturl foods s well s on rtificil diets (Simpson nd Rubenheimer, 1993; Rubenheimer nd Simpson, 1999). Prsitism by C. congregt significntly ltered the reltive mounts of the crbohydrte nd protein diets consumed by host lrve. Conditioning newly moulted prsitized nd norml lrve on either the crbohydrte diet or the protein diet before offering the dietry choice hd limited long-term effects on subsequent diet consumption. The results of these experiments, however, must be interpreted with cre, s both conditioning nd prsitism independently ffected the llometric reltionship between lrvl size nd consumption. We know of no other investigtions demonstrting this type of size-relted phenomenon regrding conditioning nd prsitism. short-term effect of conditioning clerly ws evident in lrve conditioned on the protein diet. When offered choice between the diets, lrve selected nd fed on the crbohydrte diet for severl hours rther thn on the protein diet. These results re consistent with those of Friedmn et l. (1991), who lso observed short-term feeding on sucrose diet selected by H. ze conditioned on high csein diet. Our results were lso consistent, in prt, with those reported by Simpson et l. (19)

12 76 S. N. Thompson, R.. Redk nd L.-W. Wng Glutmte C5 C Gl x C2 Trehlose C1 Glutmine C5 lnine C2 C5 + glycerol C2 C3 Citrte C3 Trehlose lnine lnine C3 Gl x C C2 + glycerol phosphte C2 Mlte C C (p.p.m.) Fig. 7. Typicl 13 C NMR spectrum of perchloric cid extrcts of hemolymph from fifth-instr M. sext lrve given dietry choice for h nd dministered [2-13 C]pyruvte. () Spectrum in the region 5. p.p.m. to 21 p.p.m. showing enrichments for lnine, trehlose nd C5 of glutmte nd glutmine. () Prtil spectrum in the region 1 96 p.p.m. showing 13 C-enrichments for C2 nd C3 of lnine nd Gl x. (C) Prtil spectrum in the region p.p.m. showing the selective 13 C-enrichment in trehlose, s well s other C Internl stndrd C6 Serine C2 Tble 6. NCOV summry demonstrting the effects of prsitism on the hemolymph trehlose level nd glucogenic formtion of trehlose by the host Mnduc sext, given dietry choice (see Tble 5) d.f. Men squre F P Experiment 1 Protein/crbohydrte diet consumption rtio Prsitism =.115 Initil mss (covrite) =.721 Error G1 x C2/C3 13 C-enrichment rtio Prsitism =.27 Initil mss (covrite) =.3122 Error lnine C3/C2 13 C-enrichment rtio Prsitism =.2 Initil mss (covrite) =.973 Error trehlose C6/lnine C3 13 C-enrichment rtio Prsitism =.6679 Initil mss (covrite) =.7697 Error [Trehlose] (mmol l 1 ) Prsitism =.972 Initil mss (covrite) =.73 Error Experiment 2 Protein/crbohydrte diet consumption rtio Prsitism =.51 Initil mss (covrite) =.15 Error G1 x C2/C3 13 C-enrichment rtio Prsitism =.96 Initil mss (covrite) =.762 Error lnine C3/C2 13 C-enrichment rtio Prsitism =.6 Initil mss (covrite) =.352 Error trehlose C6/lnine C3 13 C-enrichment rtio Prsitism =.1 Initil mss (covrite) =.257 Error [Trehlose] (mmol l 1 ) Prsitism =.161 Initil mss (covrite) =.33 Error 7 metbolites, including glycerol nd glycerol phosphte with the sme or similr chemicl shifts. Hemolymph ws collected 3.5 h postinjection. The spectrum ws generted from dt cquisitions nd is shown with 3 Hz line brodening. See text for explntion of the metbolic derivtion of 13 C-enrichments.

13 Dietry intke nd metbolic homeostsis in M. sext 77 Hemolymph Ft body CO 2 Gluconeogenesis COO CO~P CH 2 Trehlose C 1 C 2 C 3 C C 5 C 6 Phosphoenolpyruvte Pyruvte COO C=O CH 3 CO Pyruvte 2 COO C=O CH 3 Oxlocette Fumrte CO 2 TC cycle Glycolysis CO2 cetyl Co CO 2 COO H CNH + 3 CH 2 CH 2 H 2 N CO lnine COO H CNH + 3 CH 3 Fig.. Metbolism of [2-13 C]pyruvte by fifth-instr M. sext lrve, illustrting the derivtion of 13 C-enrichment in glycolytic nd gluconeogenic intermedites, nd in hemolymph trehlose. Principl metbolic derivtions re shown with different symbols. Circles, 13 C enrichments derived directly following crboxyltion of the originl 13 C in [2-13 C]pyruvte, s dministered to lrve; * 13 C-enrichments derived from [2-13 C]pyruvte, but following crboxyltion nd rndomiztion t the fumrse-ctlyzed step of the TC cycle; 13 C-enrichments lso derived from [2-13 C]pyruvte following decrboxyltion to cetyl-coenzyme, condenstion with oxlocette nd subsequent synthesis of Gl x. The metbolic effects of pentose cycling re not shown. from short-term experiments with lrve of Spodopter exigu (Hubner), nother lepidoptern insect. In tht study, insects were conditioned for 12 h on csein diet, nd subsequently given the choice of csein or sucrose diet. Lrve consumed significntly more of the crbohydrte diet over 12 h feeding period. When S. exigu lrve were conditioned in the sme mnner, but on the sucrose diet, nd were then presented with choice of the diets, they not only fed more on the protein diet, but consumed significntly less of the crbohydrte diet thn did lrve tht were conditioned on nutritionlly complete diet contining both csein nd sucrose. lthough the mount of diet consumed by M. sext during the present short-term selection experiments ws not determined, our observtions on the proportion of lrve choosing ech diet following the conditioning period indicte tht lrve conditioned on the sucrose diet selected in mnner similr to lrve tht were not conditioned, nutritionl stte tht my be comprble to tht of lrve conditioned on nutritionlly complete diet. Findings from the short-term dietry selection experiments with M. sext lrve suggest n explntion for the difference in the results observed between the duplicte long-term selection conditioned experiments. In Experiment 2, norml lrve conditioned on the protein diet consumed smll but significntly greter mount of the crbohydrte diet thn lrve conditioned on the crbohydrte diet (Fig. 2, Tble 2). Moreover, these lrve displyed significntly greter finl mss. The length of time tht lrve initilly feed on the sucrose diet fter being conditioned on the csein diet my ffect the totl mount of crbohydrte nd protein consumed over the entire 2-dy feeding period. The totl mount of crbohydrte consumed my be significntly reduced, while the mount of the protein diet consumed my increse. The ltter effect could resonbly result in incresed growth. Previous studies suggest tht this initil feeding period is highly vrible nd my rnge from few to severl hours (Friedmn et l., 1991; Simpson et l., 19; Thompson nd Redk, 2). In the present short-term experiments, for exmple, the mjority of norml lrve tht fed on the sucrose diet fter being conditioned on the csein diet begn to switch nd feed on the protein diet fter pproximtely 2.5 h (Fig. ). Most prsitized lrve did not chnge diets during the h observtion period (Fig. ). Studies by Friedmn et l. (1991) indicte tht with H. ze the length of this period is relted to hemolymph trehlose level. fter conditioning on csein diet lcking sucrose, lrve fed on sucrose diet for pproximtely 3 h, nd then they begn to chnge over nd feed on the csein diet. nlysis of hemolymph trehlose demonstrted tht lrve hve very low trehlose level fter conditioning on the csein diet, nd upon switching to the crbohydrte diet trehlose levels increse nd norml levels re reched in pproximtely 3 h. The ltered rtio of protein/crbohydrte diet consumed by prsitized M. sext lrve resulted principlly from decrese in protein, rther thn chnge in crbohydrte consumption. s prsitized lrve consumed rtio of protein/crbohydrte pproching 1:1 in most of the experiments, it is importnt to consider whether the results reflect rndom unregulted feeding or n ltertion in the intke trget. The present study is equivocl in this regrd. While the short-term experiments clerly indicted the bility of prsitized insects to select between diets of different nutritionl content following the conditioning period, this result does not definitively estblish n ltered food preference by prsitized lrve. To clerly estblish whether prsitism induces n ltered intke trget, lrve must be nutritionlly chllenged by being provided with choices of diets hving vriety of nutrient rtios, nd documented to defend the sme intke trget (Rubenheimer nd Simpson, 1999). lck in the defense of n intke trget by lrve would indicte rndom feeding. In the cse of prsitized M. sext, rndom feeding could reflect