Nutrition interacts with parasitism to influence growth and physiology of the insect Manduca sexta L.

Transcription

1 The Journl of Experimentl iology 28, Published by The ompny of iologists 25 doi:1.1242/jeb Nutrition intercts with prsitism to influence growth nd physiology of the insect Mnduc sext L. S. N. Thompson, R.. Redk nd L.-W. Wng Deprtment of Entomology, University of liforni, Riverside, liforni 92521, US uthor for correspondence (e-mil: ccepted 24 November 24 The influence nd interction of dietry protein:crbohydrte blnce nd prsitism by otesi congregt on nutrient intke nd growth were exmined over the lst two lrvl stdi of Mnduc sext. Effects of nutritionl sttus on host blood metbolite concentrtions were lso determined. Six ft-free chemiclly defined diets were tested, ech hving the sme totl level of csein nd sucrose, but with csein to sucrose rtios vrying from low protein/high crbohydrte to equl levels of both nutrients through to high protein/no crbohydrte. Nutrient rtio nd prsitism ech ffected nutrient consumption nd growth. Feeding responses differed between norml nd prsitized lrve, s illustrted by nutrient rrys, two-dimensionl plots of protein nd crbohydrte consumption on diets hving different nutrient rtios. Norml lrve consumed more nutrients nd took longer to develop s dietry nutrient rtio ws displced from equl levels of both nutrients. Except on the diet hving the sme mount of protein nd crbohydrte, prsitized lrve consumed less nutrients thn norml lrve, lthough on ll diets prsitized lrve took longer to develop. When the contribution of prsite biomss ws excluded, prsitized lrve showed lower mss gin thn norml lrve on ll diets. Totl mss gin by norml nd prsitized lrve with prsite biomss included, however, ws similr on diets hving intermedite nutrient rtios. Differences in mss gin between diets reltive to nutrient consumption were evident from multidimensionl representtions of mss gin with protein nd crbohydrte consumption. Three-dimensionl plots nd contour mps of norml nd prsitized lrve were different. When differences in nutrient consumption between diets were tken into ccount, protein Summry consumption hd greter effect on growth thn crbohydrte consumption nd norml lrve generlly displyed greter mss gin thn prsitized lrve on the sme diets. Utiliztion efficiency, the efficiency of conversion of ingested food to body mss, ws, therefore, generlly reduced in prsitized insects. oncentrtions of blood protein, totl free mino cids nd trehlose were ech influenced by dietry nutrient rtio nd prsitism. oncentrtions of protein nd free mino cids generlly incresed nd trehlose concentrtion decresed s dietry protein incresed nd crbohydrte decresed. The opposite ws the cse s dietry crbohydrte incresed nd protein decresed. Dietry nutrient rtio, however, ffected norml nd prsitized lrve differently. Prsitized lrve hd higher overll trehlose concentrtions while norml lrve hd higher protein nd totl free mino cid concentrtions. When differences in nutrient consumption between diets were ccounted for, protein consumption hd greter effect on blood protein nd free mino cid concentrtions thn did dietry nutrient rtio or prsitism. Protein consumption, however, did not ffect trehlose concentrtion. rbohydrte consumption hd no effect on the concentrtion of ny of the metbolites fter differences in nutrient consumption were tken into ccount. Effects of nutrient consumption on trehlose concentrtion, therefore, were due to dietry nutrient rtio nd prsitism. The potentil relevnce of the bove findings to the biology of prsitized M. sext lrve is discussed. Key words: insect, growth, nutrition, diet, prsitism, Mnduc sext, otesi congregt. Introduction Nutrition is the essentil connection between n niml nd its environment, providing the energy nd the chemicls necessry for survivl. nimls cquire nutrients through n integrtion of behvior nd physiology tht is considered the subject of nutritionl ecology (House, 1977; Slnsky, 1982). This integrtion ensures tht n optiml quntity nd blnce of the required nutrients re consumed nd vilble for growth, development nd reproduction (Simpson nd Rubenheimer, 1993; Rubenheimer nd Simpson, 1999). During multitrophic interctions, nourishment of predtor or prsite is chieved by prior provision of nutrients to herbivorous hosts nd prey. dditionlly, nourishment of

2 612 S. N. Thompson, R.. Redk nd L.-W. Wng mny prsites requires living host. The host must therefore consume nutrients in support of prsite growth nd development nd ensure its own survivl during tht period. In these cses, drmtic ltertions of host behvior, physiology nd development ccompny prsitism nd these ltertions often pper beneficil for prsite success (Thompson, 1993; Quickie, 1997; eckge nd Gelmn, 21). Little, however, in known of the roles of nutrition nd of nutritionl interction occurring between the host nd prsite tht my be essentil for prsite success. Importnt questions remin. How does prsitism influence host nutrition nd physiology? How do prsitism-induced ltertions of host nutrition influence prsite development? This study ddresses the first question, specificlly the effects of prsitism by gregrious brconid wsp, otesi congregt, on the feeding nd physiologicl responses of lst instr lrve of the Sphingid moth Mnduc sext, to vritions in dietry composition. These responses by prsitized host lrve will ultimtely define the nutritionl conditions under which. congregt develops. Previous studies suggest tht prsitism lters the norml feeding responses of M. sext lrve (Thompson et l., 21). Norml, unprsitized lrve given choice of two diets, one contining csein but lcking crbohydrte nd second contining sucrose but lcking protein, feed in rtio of 2 prts of the protein diet to 1 prt crbohydrte diet. Prsitized insects given the sme choice consume equl mounts of ech diet. This difference in diet consumption is principlly due to lower consumption of the protein diet by prsitized lrve, which explins in prt the slow growth of prsitized insects. When fed individul diets hving different rtios of csein nd sucrose, those prsitized lrve feeding on diet with equl mounts of protein nd crbohydrte produce the gretest number nd biomss of prsites. This suggests tht ltered host nutrient intke my be importnt for optimizing prsite growth nd development. Lcking, however, is precise informtion on the influence of dietry nutrient composition on nutrient consumption, growth nd development by norml nd prsitized lrve. Knowledge of informtion on host blood metbolite concentrtions is essentil for understnding the impct of host response to dietry nutrient rtio nd prsitism becuse these metbolites provide the nutrients on which. congregt feed, nd their concentrtions ply role in regulting feeding nd food choice by the host (Simpson nd Rubenheimer, 1993b). Nutrients digested nd bsorbed by the gut nd metbolites synthesized from nutrients nd relesed by tissues re trnsported directly in the open circultion. They occur t vrible nd often high concentrtions depending on nutrient intke. The concentrtions of circulting metbolites provide continuous reding of the insect s nutritionl nd metbolic stte, informtion tht serves s bsis for mintining nutritionl homeostsis through regultion of feeding (Schiff et l., 1989; Simpson nd Rubenheimer, 1996). Studies with severl species of lepidoptern lrve, including M. sext, demonstrte tht the concentrtion of trehlose, the blood sugr of insects, correltes positively with crbohydrte consumption. rbohydrte intke nd blood sugr level, in turn, ffect the subsequent consumption of dietry crbohydrte (Simpson et l., 1988; Friedmn et l., 1991; Thompson nd Redk, 2). Food choice is dynmic process. Mny insects offered nutritionlly unblnced foods continuously djust their feeding, selecting combintion of foods tht ultimtely results in n intke of required nutrients tht is optiml for growth nd development (Wldbuer nd Friedmn, 1991; Simpson nd Rubenheimer, 1993; Rubenheimer nd Simpson, 1999). The present study estblishes the reltionship between dietry nutrient rtio, nutrient consumption nd growth of norml unprsitized M. sext lrve nd lrve prsitized by. congregt. We fed lrve series of chemiclly defined diets ll contining the sme totl level of csein nd sucrose but with vrible rtios of these nutrients. We lso exmined the effects of dietry nutrient rtio nd nutrient intke on the blood concentrtions of protein, totl free mino cids nd trehlose. sed on the results of erlier studies described nd cited bove, we predicted tht both dietry nutrient level nd prsitism would ffect diet nd nutrient consumption. Further, we predicted tht the levels of blood metbolites would reflect the intke of csein nd sucrose by lrve fed the vrious diets. Lst, we consider how the effects nd interctions of nutrition nd prsitism my influence the feeding behvior nd ecophysiology of M. sext lrve. In subsequent study we will exmine how these effects of prsitism directly influence prsite growth nd development. Mterils nd methods Insect culture Stock colonies of Mnduc sext L. were rered on semidefined rtificil diet contining whet germ, Torul yest, linseed oil, Wesson s inorgnic slt mixture, scorbic cid nd -complex vitmins (ell nd Jochim, 1976). Following egg htching, first instr lrve were housed individully in multiwell tissue culture pltes nd trnsferred to lrger plstic continers s they grew nd developed. Upon completion of lrvl development, fifth or terminl stge lrve were removed from the diet continers nd plced in holes bored in smll wooden blocks where they pupted. dult moths mted nd lid eggs on greenhouse tomto plnts. Mixed-sex popultions of pproximtely 1 15 otesi congregt Sy dult prsitoids were housed in 3 l glss jrs. M. sext lrve in the second instr were plced in the bottom of the jrs nd superprsitized two to three times. The prsitized host lrve were removed, plced on rtificil diet, nd treted in the sme fshion s norml lrve described bove. Prsitized host lrve did not pupte nd prsitoids emerged from hosts in the terminl stdium housed in 16 ml plstic cups. Prsitoid cocoons were crefully removed from the surfce of hosts nd plced in 3 ml plstic cups, pproximtely 15 per cup. ups were then individully plced in 3 l glss jrs for emergence. fter eclosion dult prsitoids

3 Nutrition nd prsitism ffect growth of M. sext 613 were fed honey nd supplied with wter from dentl cotton wicks in smll test tubes. Insects were mintined in Precision Scientific incubtor t 28 with 16 h:8 h light:drk non-dipusing, long-dy photocycle. Experimentl rering protocol Norml nd prsitized lrve were fed chemiclly defined rtificil diet (hmd et l., 1989) immeditely upon moulting to the fourth stdium. The diet contined csein nd sucrose s digestible protein nd crbohydrte, respectively. The stock diet formultion contined 9 g l 1 of ech nutrient. In ddition, the diet consisted of linseed oil, Wesson s slts, scorbic cid nd vitmins. Nutrients were principlly obtined from Nutritionl iochemicls (levelnd, OH, US) nd ioserve (Frenchtown, NJ, US). Experiments were conducted with series of six diets, ech with n equivlent mount of combined protein nd crbohydrte, but with the following rtios of csein to sucrose:.125:1.875s,.25:1.75s,.5:1.5s, 1.:1.S, 1.5:.5S nd 2.:S. The vlues re reltive to the mount of csein nd sucrose in the stock formultion, tht is 1:1S is equivlent to 9 g l 1 csein nd 9 g l 1 sucrose. The nutrient content of the diets were equivlent in mss, nd becuse crbohydrte nd protein hve similr cloric vlue the diets were pproximtely equl in energy content. Non-feeding phrte fourth instr lrve were removed from the rering diet nd superprsitized two to four times, s described by lleyne (1995). This produced high prsite burdens or mximl numbers of prsites developing within individul host lrve. Lrve were synchronized s described by ker et l. (1987). In our studies, however, prsitized insects were not strved for 1-12 h fter moulting, but immeditely plced on the experimentl diets. Groups of ten rndomly selected prsitized lrve were mintined on ech of the experimentl diets for the feeding studies described below. Norml fourth instr lrve t the equivlent stge of development served s controls. ll lrve were housed individully in 3 ml plstic cups during the fourth stdium, nd phrte fifth instr lrve were trnsferred to 16 ml cups for the durtion of the experiment. Insects were mintined t 28 with long-dy photocycle s described bove. Feeding studies Lrve were fed the vrious experimentl diets throughout the fourth stdium until the end of the fifth stdium. In the cse of norml lrve, the experiments were discontinued once fter pproximtely 25% of the lrve hd stopped feeding nd entered the wndering phse in preprtion for puption. Norml lrve wnder within 6 12 h of ech other nd development time on the vrious diets ws mesured in full dy increments. t this point, ll lrve hd reched fresh mss of pproximtely 8 1 g with totl development time between 7 nd 15 dys, depending upon diet. In the cse of prsitized lrve, cesstion of feeding occurred over longer time intervl, up to 24 h. The time lso vried with diet, between 12 nd 15 dys. Determintion of nutrient consumption nd lrvl growth Diet consumption ws mesured s the difference between the totl mount of diet offered to lrve nd the mount remining in the diet cups t the end of the experiment together with undigested diet remining in the gut. Dry mss of the diet remining in the cups ws determined by drying the diet in n oven t 1 for 24 h nd weighing on microblnce. Initil dry mss of the diet offered to the insects ws estimted from the known rtio of wet/dry mss. Protein nd crbohydrte consumption were estimted bsed on the composition of ech diet. Fecl mteril, collected t the end of the experiment, ws lso dried nd weighed. The mount of food ssimilted ws clculted s the difference between the food consumed nd the mount of frss produced on the different diets. The guts of norml nd prsitized lrve were dissected t the end of the experiment nd the diet remining in the gut ws removed. This diet ws dded to tht remining in the diet cups, for estimtion of diet consumption. The insect crcsses, including the gut, were dried s described bove, fter which they were removed nd weighed. Initil dry mss of lrve ws estimted from the wet mss nd the wet/dry mss rtio of severl lrve tht were dried t the beginning of the experiments. nlysis of host blood metbolites nlyses were conducted to determine the effects of dietry nutrient rtio nd prsitism on blood concentrtions of trehlose, protein nd totl free mino cids. Groups of five lrve were mintined on ech of the diets until pproximtely two dys before feeding stopped. The time chosen ws bsed on the results for the feeding studies described bove. We did not wit until feeding hd stopped, in order to void difficulties in collecting blood t the lter time, rpid blood cogultion in the cse of norml lrve nd interference from prsites in prsitized lrve. lood ws collected from smll incisions mde with microscissors in one or more prolegs. To remove blood cells nd tissue debris, whole blood ws centrifuged t 3 g for 3 min in refrigerted eckmn microfuge. Trehlose ws determined by 13 nucler mgnetic resonnce (NMR) spectroscopy following deproteintion of the cell-free superntnt by ddition of perchloric cid to 3.5%. Following centrifugtion s bove, blood plsm ws neutrlized with 2 M K 2 O 3. Smples were refrigerted for pproximtely 12 h nd then centrifuged to remove KlO 3. n internl stndrd, 3-(trimethylsilyl)-1-propne sulfonic cid, ws dded to the superntnt to 2 mmol l 1. Deuterium oxide, dded to 15%, served s field frequency lock. 13 NMR nlysis ws conducted under non-sturting conditions t MHz in Vrin Inov 3 spectrometer s described previously (Thompson et l., 22). Spectr were generted from 4 dt cquisitions nd referenced to the internl stndrd t pproximtely 2.67 ppm. Trehlose concentrtion

4 614 S. N. Thompson, R.. Redk nd L.-W. Wng ws clculted by compring the signl intensity of trehlose 1 t 93 ppm with the intensity of the internl stndrd. For nlysis of protein nd mino cids, blood plsm ws deproteinized by ddition of sulphoslicylic cid to 2%. fter centrifugtion, the protein pellet ws dried for 2 h t 1 nd the crude protein determined grvimetriclly with Srtorius M2P electronic microblnce (Goettingen, Germny). mino cid nlysis ws conducted with eckmn 63 utomted mino cid nlyzer (Fullerton,, US), equipped with lithium ion exchnge column. Smples were diluted 5 fold with eckmn Li-S high performnce lithium citrte buffer contining 5 µmol l 1 minoethyl cysteine s n internl stndrd. Smple liquots of 5 µl were loded into the nlyzer. mino cid seprtions were chieved by stepwise ph elution with lithium citrte buffers: ph 2.92, 3.65 nd (Li292, Li365 nd Li375, respectively; Pickering Lbortories, Mountin View,, US). vrible temperture progrm of 32, 5 nd 7, respectively, ws employed upon ddition of ech elution buffer. ninhydrin rection ws used for detection. Totl free mino cid concentrtion ws clculted by summing the concentrtions of the individul mino cids. oncentrtions of ll blood metbolites re reported in mg ml 1 blood plsm. Generl sttisticl nlyses Two-wy nlysis of vrince (NOV) principlly ws used to nlyze dt from the feeding experiments to estblish the effects of dietry nutrient rtio nd prsitism on host growth (mss gin) nd blood metbolite levels. Owing to potentil vrition in initil mss between tretments, we first nlyzed the growth dt by nlysis of covrince (NOV), using host initil mss s the covrite (Rubenheimer nd Simpson, 1992; Horton nd Redk, 1993). There ws no initil mss covrite effect, nd dt were subsequently exmined by NOV, with NOV pplied in prticulr cses where described. Normlity nd homogeneity of vrince were estblished with the Shpiro- Wilk W test nd norml probbility plots. ll dt met the ssumptions of nlysis of vrince. Results Effects of dietry nutrient rtio nd prsitism on nutrient consumption nd development time Dietry nutrient rtio nd prsitism ech ffected nutrient consumption. ecuse intke of protein nd crbohydrte vried in fixed proportions equl to nutrient rtio, we exmined nutrient intke, ccording to the method of Rubenheimer nd Simpson (1999, 23). Using this pproch, bivrite plot simultneously illustrtes protein nd crbohydrte consumption (Fig. 1). single point in two-dimensionl spce indictes the intke of the two nutrients by lrve on ech diet. Ech point lies on line or nutrient ril with ril ngle (slope of the line) equivlent to the nutrient rtio of tht diet. The reltionship between the intke points for ll diets is n intke rbohydrte consumption (g) :1.875S.25:1.75S (15) (14) (12) (12) (14) (7) (15) (7) (12).5:1.5S (12) (12) (9) 1 2 Protein consumption (g) 1.:1.S Norml Prsitized 1.5:.5S 2.:.S 3 4 Fig. 1. Two-dimensionl representtion of nutrient intke by norml unprsitized M. sext lrve (filled circles), nd lrve prsitized by. congregt (open circles), mintined over the fourth nd fifth stdi on chemiclly defined rtificil diet hving vrying rtios of csein nd sucrose. Dotted lines re nutrient rils representing the trjectory of nutrient intke expected for lrve feeding on the individul diets. Nutrient intke points indicte ctul nutrient intke (± S.E.M.). Significnt differences (P<.5) in nutrient intke between norml nd prsitized lrve on the sme diet re indicted by n sterisk. The lines through individul intke points for norml nd prsitized lrve re the intke rrys. Development times for ech dietry nutrient rtio re shown in prentheses. Dietry nutrient rtios re shown reltive to the level of ech nutrient (, csein; S, sucrose) in the bsl chemiclly defined formultion where 1.=9 g l 1. rry reflecting the feeding response to vritions in nutrient blnce s well s ny differences in development time. The nutrient intke trget is the mount nd blnce of nutrients required by the insect for optiml growth nd development over the feeding period investigted. Owing to post-ingestive effects, tht is, physiologicl mechnisms involved in processing nutrients fter ingestion, insects my disply ner optiml growth nd development under conditions where the nutrient intke trget is not met (Rubenheimer nd Simpson, 1999; see below). Norml nd prsitized M. sext lrve displyed distinct intke rrys over the fourth nd fifth stdi (Fig. 1). Differences in development time between diets, described below, contribute gretly to the overll nutrient intke ptterns for both norml nd prsitized insects. The intke rry of

5 Nutrition nd prsitism ffect growth of M. sext 615 prsitized lrve ws non-liner with the ends curved shrply inwrds demonstrting reduced nutrient intke t more extreme dietry nutrient rtios. The nutrient intke rry for norml lrve generlly formed liner configurtion. The intke point for the.125:1.875s diet, however, ws rdiclly displced from the overll rry. lerly, lrve were unble to djust their feeding response to ccommodte the low protein nd nutrient imblnce of this diet nd, therefore, the diet cn be considered pthologicl food (Rubenheimer nd Simpson, 1999). Totl nutrient intke for the vrious diets ws mesured by the distnce from the origin of the intke points long the nutrient ril of ech diet clculted from protein nd crbohydrte consumption using Pythgors s theorem (Rubenheimer nd Simpson, 23). Differences in nutrient intke between norml nd prsitized lrve were estblished using t-tests (ssuming unequl vrinces), compring the distnces long the individul nutrient rils. Norml lrve consumed significntly more of the nutrients on the.25:1.75s diet thn did prsitized lrve (t=7.86, P<.1), while prsitized lrve consumed more nutrients on the 1.:1.S diet (t= 3.97, P=.19; Fig. 1). Dietry nutrient rtio ffected development time (Fig. 1). Norml lrve displyed longer development times t the extreme dietry nutrient rtios, especilly on diets hving lesser mounts of protein. sttisticl nlysis ws not possible becuse feeding of ll lrve on the individul diets terminted t the sme time. Exempting the.125:1.875s diet, incresed development time ws ssocited with incresed nutrient consumption by norml lrve over the lst stdi. Prsitized lrve showed similr but less severe trend for development time. Nutrient consumption for prsitized lrve on the higher protein diets incresed shrply when compred with norml lrve. In contrst to the results with norml lrve, however, higher nutrient consumption by Tble 1. NOV summry demonstrting the effects of dietry nutrient rtio nd prsitism by otesi congregt on host growth (mss gin), over the fourth nd fifth stdi of Mnduc sext lrve mintined on chemiclly defined rtificil diet Men Dependent vrible d.f. squre F vlue Probbility Totl host mss gin Dietry nutrient rtio <.1 Prsitism <.1 Interction <.1 Error 7.36 Host mss gin (prsites excluded) Dietry nutrient rtio <.1 Prsitism <.1 Interction <.1 Error prsitized lrve ws not pprent with incresed development time on lower protein diets. Effects of dietry nutrient rtio nd prsitism on host growth nd utiliztion efficiency Lrvl mss gin, with or without prsite biomss included, ws ffected by dietry nutrient rtio nd prsitism (Tble 1). Mss gin ws gretest on the.5:1.5s, 1.:1.S nd 1.5:.5S diets, nd ws less on the other diets hving more extreme nutrient rtios (Fig. 2). There were significnt interctions between dietry nutrient rtio nd prsitism, demonstrting tht dietry nutrient rtio ffects mss gin differently in norml nd prsitized lrve (Tble 1). When prsite biomss ws included s mss gin, there ws no difference in mss gin between norml nd prsitized lrve on the 1.:1.S nd the 1.5:.5S diets, but on the other diets, mss gin ws significntly less for prsitized insects (Fig. 2). Host mss gin (g) 1..5 Norml Prsitized Prsitized (excluding prsites) c D,D.125:1.875S.25:1.75S.5:1.5S 1.:1.S 1.5:.5S 2.:S Dietry nutrient rtio b Fig. 2. Effects of dietry nutrient rtio nd prsitism by. congregt on growth (mss gin) of M. sext lrve mintined over the fourth nd fifth stdi on chemiclly defined rtificil diet hving vrying rtios of csein nd sucrose. rs show mens ± S.E.M. Significnt differences (P<.5) mong norml lrve re indicted by different lowercse letters nd mong prsitized lrve (including prsite biomss) by different upper cse letters. Significnt differences between norml nd prsitized (including prsite biomss) lrve on the sme diet re indicted by n sterisk. Dietry nutrient rtios re shown reltive to the level of ech nutrient (, csein; S, sucrose) in the bsl chemiclly defined formultion where 1.=9 g l 1.

6 616 S. N. Thompson, R.. Redk nd L.-W. Wng Excluding the prsite contribution, the mss gin by prsitized insects ws less thn tht of norml lrve on ll the diets (Fig. 2); n effect most pronounced on the most suitble diets bove. Further, prsitism hd much greter impct on host mss gin thn did dietry nutrient rtio when prsite biomss ws excluded (s estimted by men squre vlues; Tble 1). To estblish whether dietry nutrient rtio nd prsitism ffected mss gin independent of nutrient consumption, we conducted n nlysis of mss gin by two-wy NOV using protein nd crbohydrte consumption s joint covrites. The nlysis confirmed the importnce of protein nd crbohydrte for mss gin, s there ws significnt effect of ech nutrient covrite (Tble 2). Protein consumption hd the gretest influence on totl mss gin tht included prsite biomss. Without prsite biomss considered, prsitism hd the lrgest effect on mss gin, but of the two nutrient covrites, protein hd greter effect thn crbohydrte. There were no interctions involving either nutrient covrite with dietry nutrient rtio or prsitism. Hving ccounted for differences in nutrient consumption, the effects of dietry nutrient rtio nd prsitism on host mss gin re due to ltered utiliztion efficiency, the efficiency of conversion of food consumed to body mss. There ws lso n interction between dietry nutrient rtio nd prsitism on mss gin, demonstrting tht the effect of dietry nutrient rtio on mss gin differs between norml nd prsitized lrve. Tble 2. NOV summry demonstrting the effects of dietry nutrient rtio nd prsitism by otesi congregt on host growth (mss gin), over the fourth nd fifth stdi of Mnduc sext lrve, fter ccounting for differences in nutrient consumption between diets Men Dependent vrible d.f. squre F vlue Probbility Totl host mss gin Dietry nutrient rtio <.1 Prsitism <.1 Interction =.8 ovrite (protein <.1 ovrite (crbohydrte <.1 Error Host mss gin (prsites excluded) Dietry nutrient rtio <.1 Prsitism <.1 Interction <.1 ovrite (protein <.1 ovrite (crbohydrte <.1 Error 68.5 Dietry nutrient rtio nd prsitism were min tretment effects nd protein nd crbohydrte consumption were joint covrites. Prsitized lrve displyed lesser totl mss gin, nd thus lower utiliztion efficiency, thn norml lrve on ll diets except the.125:1.875s nd.25:1.75s diets where the mss gin of norml nd prsitized lrve ws similr (Fig. 3). When prsite biomss ws excluded, prsitized lrve grew less thn norml lrve on ll diets except the.125:1.875s diet. With or without prsite biomss considered, the nlysis ccounted for pproximtely 97% of the vrition within the dt. Multi-dimensionl profiles showing the reltionship between mss gin nd nutrient intke indicte how nutrient consumption ffects growth of norml nd prsitized M. sext lrve (Fig. 4). Three-dimensionl plots of mss gin with protein nd crbohydrte consumption were modeled using SS (PRO GRID followed by PRO RSREG nd PRO G3D. SS version 8.2, 21, SS Institute Inc. ry, N, US). This methodology uses lest-squres pproch to fit qudrtic response surfce regression model utilizing protein nd crbohydrte consumption s independent vribles nd lrvl mss gin s dependent vrible (mss gin = protein consumption 2 + crbohydrte consumption 2 + [protein consumption crbohydrte consumption] + protein consumption + crbohydrte. dditionlly, contoured surfce mps were generted (PRO GONTOUR) from 4 point mtrix creted by interpolting simple liner function for the reltionship between dietry nutrient intke nd estimted mss gin by norml nd prsitized lrve (PRO G3GRID). For the nlysis of prsitized insects on the 1.5:.5S diet, we deleted the result for one lrv tht hd biomss twice tht of the others in this group. The contribution of this lrv resulted in stndrd error for mss gin of twice tht of ny other diet group (Fig. 2). Three-dimensionl models show the nutrient spce supporting growth of norml nd prsitized insects (Fig. 4). The model for norml unprsitized lrve ccounted for pproximtely 96%, nd for prsitized lrve 95%, of the vrition within the dt. Growth of norml lrve is greter over broder rnge of nutrient intke. The effects of nutrient consumption re generlly consistent with mss gin (Fig. 2) nd nutrient consumption (Fig. 1) by lrve on the vrious diets. lthough growth of both norml nd prsitized lrve is very low t low levels of protein consumption (Figs 1, 2), the model incorrectly suggests growth of prsitized lrve in the bsence of protein consumption. The models, however, confirm tht mximl growth of norml nd prsitized lrve occur when protein nd crbohydrte re consumed in equl mounts, pproximtely 2 g of ech nutrient. The three-dimensionl model for prsitized lrve ws similr whether prsite biomss ws included or excluded (not shown) from the nlysis, except mss gin ws lower without prsite contribution. The models re only predictive within the rnge of ctul nutrient consumption pproximtely.1 2. g csein nd 4.6 g sucrose for norml lrve nd g csein nd 2.9 g sucrose for prsitized lrve. The contour mps demonstrte more precisely the growth expected for nutrient intke by lrve on specific diets. It is cler tht the mximl growth of norml lrve occurs on the.25:1.75s,.5:1.5s, 1.:1.5S nd 1.5:.5S diets, while growth of prsitized lrve

7 Nutrition nd prsitism ffect growth of M. sext 617 Host mssgin (g) Norml Prsitized Prsitized (excluding prsites) c,d b,c.125:1.875s.25:1.75s.5:1.5s 1.:1.S 1.5:.5S 2.:S,b Dietry nutrient rtio c d Fig. 3. Effects of dietry nutrient rtio nd prsitism by. congregt on growth (mss gin) of M. sext lrve mintined over the fourth nd fifth stdi on chemiclly defined rtificil diet hving vrying rtios of csein nd sucrose, fter ccounting for differences in protein nd crbohydrte intke between diets. rs show lest-squre mens ± S.E.M. Differences between diets nd between norml nd prsitized lrve re due to differences in utiliztion efficiency. Significnt differences (P<.5) mong norml lrve re indicted by different lowercse letters nd mong prsitized lrve (including prsite biomss) by different upper cse letters. Significnt differences between norml nd prsitized (including prsite biomss) lrve on the sme diet re indicted by n sterisk. Dietry nutrient rtios re shown reltive to the level of ech nutrient (, csein; S, sucrose) in the bsl chemiclly defined formultion where 1.=9 g l 1. Host mss gin (g) Host mss gin (g) Protein consumption (g) Protein consumption (g) rbohydrte consumption (g) 4 5 rbohydrte consumption (g) rbohydrte consumption :1.875S :1.75S 1.3.5:1.5S 1.5:.5S 2.:S :1.875S D :1.75S Protein consumption :1.5S :1.S 1.:1.S :.5S 2.:S Fig. 4. Multi-dimensionl profiles illustrting the effects of nutrient consumption on growth (mss gin) by norml unprsitized M. sext lrve nd lrve prsitized by. congregt, mintined over the fourth nd fifth stdi on chemiclly defined rtificil diet hving vrying rtios of csein nd sucrose. (,) Three-dimensionl models for norml nd prsitized lrve. () Norml: F=49.4, P<.1, r 2 =.9491; () prsitized: F=148.38, P<.1, r 2 = (,D). ontour mps for norml nd prsitized lrve, respectively. Nutrient rils shown for the individul diets. ctul mss gin by individul lrve indicted by filled circles.

8 618 S. N. Thompson, R.. Redk nd L.-W. Wng is mximl on the 1.:1.5S diet nd to lesser degree the 1.5:.5S nd.5:.15s diets. Effects of dietry nutrient rtio nd prsitism on blood metbolite concentrtions Dietry nutrient rtio nd prsitism ech ffected the concentrtions of blood protein, free mino cids nd trehlose (Tble 3). In the cses of free mino cids nd trehlose there were significnt interctions between dietry nutrient rtio nd prsitism, indicting tht dietry nutrient rtio ffects the concentrtion of these metbolites differently in norml nd prsitized lrve. Trehlose concentrtion decresed s dietry nutrient rtio shifted from the high crbohydrte to low crbohydrte diets (Fig. 5). Only on the 1.:1.S diet ws there significnt difference between the norml nd prsitized lrve, with prsitized insects hving higher concentrtion. Over ll diets, prsitized lrve hd significntly higher trehlose concentrtion thn norml lrve, 25.21±.94 nd 2.38±.97 mg ml 1, respectively (P=.9). In contrst, free mino cids nd protein concentrtion generlly incresed s dietry nutrient rtio shifted from low protein to high protein diets. oncentrtion of free mino cids ws higher in norml lrve thn in prsitized lrve on the.25:1.75s diet nd protein concentrtion ws higher in norml lrve thn in prsitized lrve on the 2.:S diet (Fig. 5). However, protein concentrtion, over ll diets, ws higher in norml thn in prsitized lrve, 2.49±.79 nd 14.3±.79 mg ml 1, respectively (P<.1). Free mino cid concentrtion ws lso higher overll in norml lrve, 14.48±.49 nd 12.27±.5 mg ml 1, respectively (P=.28). ontour mps illustrted the effects of protein nd crbohydrte consumption on blood concentrtions of protein, free mino cids nd trehlose (Fig. 6). Profiles for nitrogen metbolites in prsitized nd norml lrve were generlly similr, but prsitized insects hd lower concentrtions t equivlent levels of nutrient consumption (Fig. 6,D nd,e). This ws most notble t high levels of protein consumption where prsitized lrve exhibited drmticlly lower blood levels of both protein nd free mino cids. Profiles for trehlose concentrtion were lso similr in norml nd prsitized lrve, but in this cse, prsitized lrve generlly hd higher trehlose concentrtions t equivlent levels of nutrient intke (Fig. 6,F). The profiles for trehlose in lood protein concentrtion (mg ml 1 ) lood free mino cid concentrtion (mg ml 1 ) d Norml Prsitized, c,d b,c,d,,b,c,,b.125:1.875s.25:1.75s.5:1.5s 1.:1.S 1.5:.5S 2.:S c D b,d b,.125:1.875s.25:1.75s.5:1.5s 1.:1.S 1.5:.5S 2.:S b b

9 Nutrition nd prsitism ffect growth of M. sext 619 lood trehlose concentrtion (mg ml 1 ) 6 4 2,b.125:1.875S.25:1.75S.5:1.5S 1.:1.S 1.5:.5S 2.:S both norml nd prsitized lrve were not s uniform s were those of protein nd free mino cid concentrtions. Trehlose mps displyed smll concentrtion foci. These reflect the lrge vrition in concentrtion mong lrve. onsequently, plots for trehlose re probbly less relible for predicting trehlose concentrtions t those levels of nutrient consumption. NOV nlyses of metbolite concentrtions using protein nd crbohydrte consumption s joint covrites, s described for growth bove, demonstrted significnt effects of dietry nutrient rtio nd prsitism in ech cse (Tble 4). There were interctions between dietry nutrient rtio nd prsitism, for trehlose nd free mino cids concentrtion but not for protein concentrtion. There ws covrite effect for protein consumption in the cses of free mino cids nd protein concentrtions, nd protein consumption hd greter effect on protein nd mino cid concentrtion thn did either dietry nutrient rtio or prsitism. No covrite effect of crbohydrte, Dietry nutrient rtio Fig. 5. Effects of dietry nutrient rtio nd prsitism by. congregt on blood concentrtion (mg ml 1 ) of () protein, () free mino cids nd () trehlose in fifth instr M. sext lrve mintined over the fourth nd fifth stdi on chemiclly defined rtificil diet hving vrying rtios of csein nd sucrose. rs show mens ± S.E.M. Significnt differences (P<.5) mong diets between norml lrve re indicted by different lowercse letters, nd between prsitized lrve (including prsite biomss) by different uppercse letters. Significnt differences between norml nd prsitized (including prsite biomss) lrve on the sme diet re indicted by n sterisk. Dietry nutrient rtios re shown reltive to the level of ech nutrient (, csein; S, sucrose) in the bsl chemiclly defined formultion, where 1.=9 g l 1.,b b c consumption ws evident for ny of the metbolites. ecuse of the bsence of significnt nutrient covrite effects for trehlose concentrtion, dietry nutrient rtio nd prsitism, rther thn differences in nutrient consumption, lrgely explin the effects of protein nd crbohydrte consumption on trehlose. There were no interctions involving the nutrient covrites with prsitism nd dietry nutrient rtio. Discussion This study demonstrtes tht dietry nutrient blnce nd prsitism interct to influence nutrient consumption nd growth of M. sext lrve. Differences in protein nd crbohydrte intke between diets resulted from the nutrient rtios of the diets nd differences in the mounts of diet consumed. Nutrient intke by norml M. sext lrve on the vrious diets ws such tht incresed consumption of one nutrient resulted in decresed consumption of the other nutrient, whether crbohydrte or protein. When linerized, this intke rry hs negtive slope < 1, demonstrting tht lrve re much more sensitive to dietry imblnces (nutritionl errors; Rubenheimer nd Simpson, 1999) in protein thn crbohydrte over the lst two stdi. In this regrd, the rry is similr to tht reported for complete lrvl development of nother lepidoptern insect, Spodopter littorlis (Lee et l., 22) nd nymphl development of two orthoptern species, Locust migrtori nd Schistocerc gregri (Rubenheimer nd Simpson, 23). Norml unprsitized lrve grow well on diets hving wide rnge of nutrient rtios where some diets hve protein nd others crbohydrte s the predominte nutrient. Of the diets tested, norml lrve hd the gretest mss gins nd shortest development times on the 1.:1.S nd 1.5:.5S diets, suggesting tht the intke trget for norml lrve lies on nutrient ril between these two. This conclusion is consistent with studies of Spodopter littorlis demonstrting tht lst instr lrve defend n intke trget hving nutrient rtio of pproximtely 1.3 prts protein to 1 prt crbohydrte (Lee et l., 22). Lrve on diets hving lower or higher protein increse their development time nd djust diet consumption in order to complete lrvl development t n pproprite size nd with sufficient reserves to pupte. Prsitism of M. sext by. congregt reduced host growth on most diets, nd lower consumption of less suitble diets ws mjor contributor to this effect. The nutrient intke rry of

10 S. N. Thompson, R.. Redk nd L.-W. Wng prsitized lrve ws shrply rced nd did not pproch linerity t the end of lrvl development s typiclly occurs with incresed development times nd nutrient consumption on unblnced diets (Rubenheimer nd Simpson, 1999). Insects were unble to djust diet consumption s the dietry nutrient rtio devited from 1.:1.S where nutrient consumption ws mximl. Thus, insects suffered lrge shortfll of deficient nutrient to void consuming smll surplus of n excessive nutrient. Prsitized lrve on unblnced diets pprently decrese their rte of feeding over time. Dietry nutrient rtio nd protein consumption re the dominnt fctors explining differences in host mss gin between norml nd prsitized lrve. Utiliztion efficiency, however, lso ffects mss gin mong diets, nd fter :1.875S.25:1.75S.5:1.5S Norml Protein (mg ml 1 ) Prsitized :1.875S.25:1.75S.5:1.5S D Protein (mg ml 1 ) :1.S :1.S :.5S :.5S :S :S rbohydrte consumption (g) :1.875S.25:1.75S.5:1.5S Free mino cid (mg ml 1 ) 1.:1.S 1.5:.5S :1.875S.25:1.75S.5:1.5S 2.5 E Free mino cid (mg ml 1 ) 1.:1.S 1.5:.5S :S :S :1.875S.25:1.75S.5:1.5S 35 Trehlose (mg ml 1 ) :1.875S.25:1.75S F.5:1.5S Trehlose (mg ml 1 ) :1.S :1.S :.5S 2.:S :.5S 2.:S Protein consumption (g) Fig. 6. ontour mps illustrting the effects of nutrient consumption on blood protein, free mino cids nd trehlose concentrtions (mg ml 1 ) during the fifth stdium of norml unprsitized fifth instr M. sext lrve nd lrve prsitized by. congregt, mintined over the fourth nd fifth stdi on chemiclly defined rtificil diet hving vrying rtios of csein nd sucrose. (,D) Protein, (,E) free mino cid nd (,F) trehlose concentrtions of ( ) norml unprsitized lrve nd (D F) prsitized lrve.

11 Nutrition nd prsitism ffect growth of M. sext 621 Tble 3. NOV summry demonstrting the effects of dietry nutrient rtio nd prsitism by otesi congregt on blood metbolite concentrtions (mg ml 1 ) in fifth instr Mnduc sext lrve mintined over the fourth nd fifth stdi on chemiclly-defined rtificil diet Men Dependent vrible d.f. squre F vlue Probbility Trehlose Dietry nutrient rtio <.1 Prsitism =.9 Interction =.13 Error Free mino cids Dietry nutrient rtio <.1 Prsitism =.28 Interction =.349 Error Protein Dietry nutrient rtio <.1 Prsitism <.1 Interction =.759 Error differences in nutrient consumption re tken into ccount, prsitized lrve generlly disply lower mss gin nd, therefore, lower utiliztion efficiency, thn norml lrve on the sme diet. For both norml nd prsitized lrve, utiliztion efficiency ws gretest on the.5:1.5s diet. sed on Wldbuer s (1968) EI (efficiency of conversion of ingested food to body substnce) nutrient rtio, the results of studies by others imply incresed utiliztion efficiency (EI) in lepidoptern lrve prsitized by otesi spp. (Slnsky, 1978; enz nd rbos, 199; lleyne, 1995). None of those studies, however, considered the influence of differences in diet or nutrient consumption between norml nd prsitized insects. We did not clculte the bove rtio, or other commonly employed nutritionl rtios becuse of difficulties inherent with their ppliction nd interprettion, first outlined by Schmidt nd Reese (1986) nd elborted by Pckrd nd ordmn (1988). NOV nlyses offer distinct experimentl s well sttisticl dvntges bsed on less restrictive ssumptions tht pply to rtio bsed prmeters (Rubenheimer nd Simpson, 1992). Numerous investigtors hve reported suppression of host growth nd food consumption by prsitized lepidoptern lrve, including M. sext (Vinson nd Iwntsch, 198; lleyne nd eckge, 1997; enz nd rbos, 199; Quickie, 1997; Nkmtsu et l., 21). The present study, however, is the first to demonstrte tht host nutrition medites prsitism effects on host growth nd consumption; whether prsitism reduces growth depends on diet. Prsitized lrve on diets hving nutrient rtios displced from the 1.:1.S nutrient rtio generlly disply reduced growth nd nutrient consumption. To chieve growth equivlent to norml lrve, prsitized lrve must consume diet hving nutrient rtio Tble 4. NOV summry demonstrting the effects of dietry nutrient rtio nd prsitism by otesi congregt on blood metbolite concentrtions (mg ml 1 ) in fifth instr Mnduc sext lrve fter ccounting for differences in nutrient consumption between diets Men Dependent vrible d.f. squre F vlue Probbility Trehlose Dietry nutrient rtio =.41 Prsitism =.21 Interction =.179 ovrite (protein =.899 ovrite (crbohydrte =.171 Error Free mino cids Dietry nutrient rtio =.21 Prsitism =.228 Interction =.119 ovrite (protein =.4 ovrite (crbohydrte =.1896 Error Protein Dietry nutrient rtio <.1 Prsitism =.32 Interction =.276 ovrite (protein =.8 ovrite (crbohydrte =.8694 Error Dietry nutrient rtion nd prsitism were min tretment effects nd protein nd crbohydrte consumption were joint covrites. close to tht of the intke trget, but lso consume more nutrients thn norml lrve. There hve been few reports of hosts with lrge numbers or burdens of otesi spp. ttining greter totl finl mss thn norml unprsitized lrve t the sme developmentl stge (Slnsky, 1978; eckge nd Riddiford, 1983; Tnk et l., 1992). We filed to observe lrger prsitized lrve under ny of the nutritionl conditions investigted during this study. Prsite biomss contributes significntly to totl host gin but the reltive contribution vries with dietry nutrient rtio. lerly, prsitism compromises host gin, but prsite biomss lone does not ccount for the difference in response of norml nd prsitized insects to dietry nutrient rtio. Incresed metbolic expenditure required to support nd sustin prsite development my ply role. Finl host mss excluding the prsite contribution is perhps the more relevnt mesure when considering potentil effects of host size on the prsite, s this is the biomss providing nourishment for

12 622 S. N. Thompson, R.. Redk nd L.-W. Wng prsite growth nd development. This issue, however, is the subject of seprte investigtion. nlysis of the host blood demonstrted the effects of dietry nutrient rtio nd prsitism on some blood metbolites. Generlly, the blood concentrtions of trehlose, free mino cids nd proteins conformed to dietry nutrient rtio. s the dietry nutrient rtio shifted towrd higher protein nd lower crbohydrte, the blood protein nd mino cid concentrtions incresed while trehlose concentrtion decresed. The effect of dietry nutrient rtio, however, differed between norml nd prsitized insects. Two explntions re generlly offered for the effects of prsitism on host tissue metbolite levels. These re tht prsitism lters the metbolic cpcity of host lrve nd chnges in metbolite levels reflect this ltertion or tht prsite bsorption nd utiliztion of host metbolites brings bout these chnges (Vinson nd Iwntsch, 198; Thompson, 1993; Nkmtsu nd Tnk, 24). lerly, the explntions re not mutully exclusive. The finding tht host ft body, the tissue regulting much of the insect s intermediry metbolism, is often diminished in prsitized lrve (Dhlmn nd Green, 1981; Nkmtsu nd Tnk, 22), including those of M. sext, strongly suggests tht the metbolic cpcity of the host is compromised. Protein synthesis, for exmple, occurs principlly in the ft body nd the overll reduction of blood protein levels observed here in prsitized M. sext is consistent with decresed ft body content. However, trehlose synthesis lso occurs in the ft body but overll trehlose concentrtions were incresed in prsitized M. sext lrve. Despite ny decrese in ft body, this results from induction of gluconeogenesis nd occurs in prsitized lrve even under nutritionl conditions tht fil to induce trehlose formtion in norml lrve (Thompson, 21; Thompson et l., 22). It my be, therefore, tht redirection of host metbolic cpcity in ddition to, or rther thn, reduction of cpcity plys n importnt role in supporting prsite growth nd development. In ny cse, the concentrtions of blood metbolites in prsitized lrve when relted to host size reflect the nutrients vilble to the developing prsite. How these fctors, host blood metbolite levels nd host size, influence prsite growth nd development is the subject of nother study. The results on blood metbolite concentrtions re equivocl regrding their potentil to influence short-term feeding preferences. In the cse of trehlose, only on the crbohydrtefree diet, 2.:.5S, were blood sugr levels low enough to suggest feeding preference for crbohydrte if these lrve were offered dietry choice of high crbohydrte nd high protein diet. Trehlose concentrtion, however, ws not different between norml nd prsitized lrve on tht diet. Effects of vritions in blood free mino cids nd protein concentrtions on feeding behvior re not known for M. sext. It is not possible t this time to predict how the vritions of these metbolites my ffect short-term feeding preferences. Investigtions with other insects, however, demonstrte reltionship between diet, blood mino cid level nd food choice (bisgold nd Simpson, 1988; Simpson nd Rubenheimer, 1993b; Znotto et l., 1996). The influence of diet nd nutrient consumption on growth of M. sext lrve determined here suggests tht norml nd prsitized insects exhibit differences in dietry bredth tht my be reflected in different long-term feeding ptterns. The intke rrys indicte tht overll, prsitized lrve exhibit less nutritionl flexibility, nd to ttin mximl host size require greter nutrient levels nd more specific nutrient blnce thn norml lrve. How this might influence the plnt feeding hbits of prsitized lrve is unknown, but prsitized lrve my prefer to feed t sites within plnts tht differ in nutrient content from those preferred by norml lrve. lterntely, the feeding by prsitized lrve my be more restrictive thn norml lrve. The results with prsitized lrve clerly suggest nrrower nutritionl preferences thn norml lrve, both, however, within the specilist ctegory. References bisgold, J. D. nd Simpson, S. J. (1988). The effect of dietry protein levels nd hemolymph composition on the sensitivity of the mxillry plp chemoreceptors of locusts. J. Exp. iol. 135, hmd, I. M., Wldbuer, G. P. nd Friedmn, S. (1989). defined rtificil diet for the lrve of Mnduc sext. Entomol. Exp. ppl. 53, lleyne, M. (1995). ltertions in host growth nd metbolism of Mnduc sext lrve prsitized by otesi congregt. MS thesis, University of liforni, Riverside,, US. lleyne, M. nd eckge, N. E. (1997). Prsitism-induced effects on host growth nd metbolic efficiency in tobcco hornworm lrve prsitized by otesi congregt. J. Insect Physiol. 43, ker, F.., Tsi, L. W., Reuter,.. nd Schooley, D.. (1987). In vivo fluctutions of JH, JH cid, nd ecdysteroid titer, nd JH esterse ctivity, during development of fifth stdium Mnduc sext. Insect iochem. 17, eckge, N. E. nd Gelmn, D.. (21). Prsitism of Mnduc sext by otesi congregt: multitude of disruptive endocrine effects. In Endocrine Interctions of Insect Prsites nd Pthogens (ed. J. P. Edwrds nd R. J Wever), pp Oxford: IOS Scientific. eckge, N. E. nd Riddiford, L. M. (1983). Growth nd development of the endoprsitic wsp pnteles congregtus: dependence on host nutritionl sttus nd prsite lod. Physiol. Entomol. 8, ell, R.. nd Jochim, R. G. (1976). Techniques for rering lbortory colonies of tobcco hornworms nd pink bollworms. nn. Entomol. Soc. mer. 69, enz, J.-. nd rbos, P. (199). Effects of dietry nicotine (.1%) nd prsitism by otesi congregt on the growth nd food consumption nd utiliztion of the tobcco hornworm, Mnduc sext. Entomol. Exp. ppl. 57, 1-8. Dhlmn, D. L. nd Green, J. R. (1981). Lrvl hemolymph protein ptterns in tobcco hornworms prsitized by pnteles congregtus. nn. Entomol. Soc. mer. 74, Friedmn, S., Wldbuer, G. P., Eertomoed, J. E., Neem, M. nd Ghent,. W. (1991). lood trehlose levels hve role in the control of dietry self-selection by Heliothis ze lrve. J. Insect Physiol. 37, Horton, D. R. nd Redk, R.. (1993). Further comments on nlysis of covrince in insect dietry studies. Entomol. Exp. ppl. 69, House, H. L. (1977). Nutrition of nturl enemies. In iologicl ontrol by ugmenttion of Nturl Enemies (ed. R. L. Ridgwy nd S.. Vinson), pp New York: Plenum. Lee, K. P., ehmer, S. T., Simpson, S. J. nd Rubenheimer, D. (22). geometric nlysis of nutrient regultion in the generlist cterpillr Spodopter littorlis (oisduvl). J. Insect Physiol. 48, Nkmtsu, Y. nd Tnk, T. (22). Lrve of n endoprsitoid, otesi