J Comp Physiol B (2008) 178:515527 DOI 10.

1007/s00360-007-0244-6

ORIGINAL PAPER

Parasitism of an insect Manduca sexta L. alters feeding behaviour and nutrient utilization to inXuence developmental success of a parasitoid
S. N. Thompson R. A. Redak

Received: 27 August 2007 / Revised: 5 December 2007 / Accepted: 16 December 2007 / Published online: 15 January 2008 Springer-Verlag 2008

Abstract The eVects of macronutrient balance on nutrient intake and utilization were examined in Manduca sexta larvae parasitized by Cotesia congregata. Insects fed an artiWcial diet having constant total macronutrient, but with varied ratios of protein and carbohydrate, with altered diet consumption in response to excesses and deWciencies of the individual macronutrients. Bivariate plots of protein and carbohydrate consumption for non-parasitized larvae demonstrated a curvilinear relationship between points of nutrient intake for the various diets, and the larvae grew best on carbohydrate-biased diets. The relationship was linear for parasitized larvae with the growth uniform across diets. On protein-biased diets, the larvae regulated the nitrogen content, containing similar amounts of nitrogen regardless of consumption. EYciency of nitrogen conversion in non-parasitized larvae was greatest on carbohydrate-biased diets, while nitrogen conversion by parasitized larvae was greatest with intermediate nutrient ratios. Accounting for carbohydrate consumption, the lipid content decreased as dietary carbohydrate increased, but parasitized larvae contained signiWcantly less lipid. The total biomass of parasites developing in individual host larvae was positively correlated with host protein consumption, but the individual parasites were similar in size. Parasitism inXuences host nutrient consumption in a manner that achieves uniform host growth under diverse nutritional regimes, thereby constraining blood nutrient concentrations within limits suitable for parasite growth and development.

Keywords Nutrient utilization Nutrition Manduca sexta Cotesia congregata Parasitism

Introduction Parasitism is the symbiotic association or durable interaction where one organism, the parasite, lives at the expense of another, the host (Roberts and Janovy 2005; Combes 2001). Parasitic associations are common among insects, involving principally insects parasitized by other insects called parasitoids (Askew 1971; Eggleton and Gaston 1990). The immature or larval stages are parasitic, while adult parasitoids are free-living. There are an estimated 600,000 species of insect parasitoids, predominantly in the order Hymenoptera, of which approximately 60,000 species are described (Eggleton and Belshaw 1992). Parasitic insects are thus among the most successful and plentiful organisms known (Hearaty 2007). The host relationships of many insect endoparasites are complex, involving dramatic changes in host behaviour and physiology (Quickie 1997) that are frequently mediated by parasitoid-derived factors including venoms and polydnaviruses, the latter an integral component of the parasitoid genome (Federici and Bigot 2003). Suppression of the host immune system (Amaya et al. 2005; Beckage 1998) and endocrine-based alterations of host development (Beckage and Gelman 2001; Edwards et al. 2001; Kelly et al. 1998) are perhaps the best characterized physiological eVects. Collectively, manifestations of parasitism often are considered manipulation of the host by the parasite (Cole et al. 2002; Jones et al. 1986; Espagne et al. 2005; Thompson 1990), a reXection of Richard Dawkins (1999) extended phenotype, where the phenotype of the parasitized host is expressed to the beneWt of the parasite rather than the host

Communicated by I.D. Hume. S. N. Thompson (&) R. A. Redak Department of Entomology, University of California, Riverside, CA 92521, USA e-mail: nelsont@ucr.edu; nelson.thompson@ucr.edu

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(Combes 2001). Other than the intuitively obvious advantage provided by immunosuppression, however, there is limited evidence in support of this paradigm of adaptive beneWt regarding the modiWed traits of parasitized insects. Indeed, the adaptive signiWcance inferred in many cases for the altered behaviour of parasitized animals fail to fulWll the minimal criteria necessary to support that conclusion (Poulin 1995). Because nourishment is essential for successful parasite growth and development, investigations of host feeding and nutrition, and of how nutrients are partitioned between host and parasite to inXuence parasite success, may provide new insights into the ultimate role and costs of parasite-induced alterations of host behaviour and physiology. Cotesia congregata is a gregarious braconid parasite of larvae of the sphingid moth Manduca sexta. Adult female parasitoids deposit large numbers of eggs into individual host larvae. After hatching, parasite larvae develop in the hosts haemocoel, feeding on the blood (haemolymph). When mature, parasitoids emerge from the host, undergo the last larval moult and subsequently form pupae from which develop adults of the next generation of parasitoids. The parasitic relationship of C. congregata with M. sexta is very suitable for examining nutritional interactions because the number of parasite larvae developing in individual host larvae is highly variable, from a few to hundreds of parasites representing in extreme cases one-quarter the entire mass of the parasite/host complex (Alleyne and Beckage 1997; Thompson et al. 2005a). Employing a geometric analysis of feeding (Simpson and Raubenheimer 1993a, b; Raubenheimer and Simpson 1997, 1999), we recently examined the eVects of dietary macronutrient ratio (protein:carbohydrate or P:C) on nutrient intake and growth over the fourth and Wfth stadia of M. sexta, comparing parasitized larvae with normal nonparasitized insects (Thompson et al. 2005a). Normal insects grew best on a diet consisting of equal amounts of protein and carbohydrate. Further, when oVered a choice of various diets having diVerent P:C ratios, these larvae consistently display an intake target ratio, the ratio of protein to carbohydrate required for optimal growth, of approximately 1:1 (Thompson and Redak 2005). When normal larvae were oVered individual diets where the P:C ratio was displaced from 1:1, insects responded by increasing or decreasing consumption to compensate for deWciencies and excesses of protein or carbohydrate in a manner predictable for a generalist feeder (Simpson and Raubenheimer 1993a, b; Thompson and Redak 2005). In contrast, parasitized insects responded to macronutrient imbalance by consuming less diet regardless of whether protein or carbohydrate was the deWcient nutrient. The total number and biomass of parasites developing in individual host larvae was correlated with host haemolymph nutrient levels (protein, amino

acids and trehalose), which, in turn, depended upon nutrient intake and dietary nutrient balance (Thompson et al. 2005a, b). A signiWcant Wnding of the above investigations was that the eVects of dietary macronutrient balance on growth and haemolymph nutrient levels in parasitized M. sexta and perhaps, therefore, the numbers of C. congregata developing in host larvae are not simply due to diVerences in nutrient intake between diets. We speculated that dietary nutrient ratio may inXuence nutrient utilization. Studies by others have established that conversion eYciency in some insects varies with dietary macronutrient balance (Raubenheimer and Simpson 1999; Zanotto et al. 1993, 1997; Lee et al. 2002). EVects of parasitism and interactions between parasitism and macronutrient balance on nutrient utilization by lepidopteran larvae are relatively unknown. While results of some investigations indicate that insect parasites themselves have high eYciencies of food utilization, these investigations failed to consider how parasite development is inXuenced by the eVects of parasitism on host nutrient intake and nutrient availability to developing parasites (Slansky and Scriber 1985; Slansky 1986; Thompson 1999). Ultimately, insect parasites obtain nourishment through the feeding activities of their hosts. Parasite development, therefore, depends on a tritrophic interaction with all trophic levels inXuencing the Wnal outcome. Furthermore, understanding how parasitism aVects host nutrient intake and utilization is critical for assessing the adaptive consequences of changes in host behaviour and physiology to successful parasite development. Here, we investigate the eVects of variation in dietary macronutrient levels on nutrient intake and utilization by normal and parasitized M. sexta larvae, and in the case of parasitized insects, distinguish nutrient utilization directed toward host mass gain from that directed towards the developing parasite. Based on earlier Wndings, we predict that changes in post-ingestive regulation of nutrient utilization occur in parasitized larvae; however, because growth of larvae is low and uniform with dietary nutrient ratio, we hypothesize that host nutrient utilization is signiWcantly reduced by parasitism and that host growth and parasite development have higher relative energy costs than is the case with growth and development of normal non-parasitized insects.

Materials and methods Insect culture M. sexta was reared on a wheat germ-based artiWcial diet (Bell and Joachim 1976), as described (Thompson and

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Redak 2005a). Second instar M. sexta larvae were parasitized by C. congregata in 3 l glass jars containing 50100 adult parasitoids of mixed sex. The parasitized host larvae were isolated and placed individually on the rearing diet. All larvae were housed in an incubator at 28C with a 16 h light/8 h dark non-diapausing, long-day photocycle. The parasites emerged from the hosts during the last or Wfth stadium. Emerging second instar parasite larvae moult into the third stadium and immediately pupate. Pupae were collected from the surface of the host and held in plastic cups, approximately 150/cup, and placed in 3 l glass jars. The emerged adult parasitoids were provided with water and honey. Rearing on experimental diets M. sexta were developmentally synchronized as pharate fourth instar larvae (Baker et al. 1987) and were superparasitized two to four times (Alleyne 1995). Larvae were superparasitized to obtain large parasite burdens, or numbers of parasites developing in individual host larvae, thereby maximizing the nutritional impact of parasitism. Normal (non-parasitized) and parasitized larvae were then maintained on the rearing diet until the end of the fourth stadium. Pharate Wfth instar larvae were placed on chemically deWned fat-free experimental artiWcial diets. The artiWcial diet was a simpliWed formulation of the chemically deWned diet described by Ahmad et al. (1989). Casein and sucrose were the sole sources of digestible protein and carbohydrate, respectively, and the basic formulation contained 60 g/l of each macronutrient. Other ingredients included: ascorbic acid, inorganic salts, cholesterol and vitamins. The nutrients were purchased principally from Nutritional Biochemicals (Cleveland, OH, USA) and Bioserve (Frenchtown, NJ, USA). The pH of the diet was adjusted with KOH to approximately 6.5. Larvae were not conditioned prior to feeding on the experimental diets and began feeding after moulting to the Wfth stadium. The experimental diets varied in protein and carbohydrate as described below and the larvae were maintained in an incubator under the conditions outlined above. Feeding protocol Groups of ten larvae were fed on one of nine experimental diets for 38 days after moulting to the Wfth stadium. The larvae were allowed to feed until cessation of feeding during the wandering phase. All diets had the same combined or total level of macronutrient, protein plus carbohydrate (120 g/l of diet), but the following ratios of casein (g/l P) and sucrose (g/l C), respectively: 100P:20C, 90P:30C, 80P:40C, 70P:50C, 60P:60C, 50P:70C, 40P:80C, 30P:90C and 100P:20C. This variation in dietary nutrient ratio nar-

rowed what was employed during our earlier investigations (Thompson et al. 2005a, b), which included extreme pathological ratios where insects failed to grow and develop. Diets were prepared in a 2% agar solution having an approximate 6.3:1 ratio of agar solution to total dry ingredients. The larvae were fed individual pieces of diet approximately 4 2.5 1 cm and weighing about 10 g in 130 ml plastic cups with snap-cap lids. They were housed in an incubator maintained at 28C. In the case of non-parasitized insects, diet consumption was measured after 2, 3 and 5 days, and at the time they stopped feeding to enter into the wandering stage in preparation for pupation. Diet consumption by parasitized larvae was similarly measured at 2, 3 and 5 days, and at the time they stopped feeding and parasite larvae began emerging. The diet was replaced as necessary. Estimation of growth and nutrient consumption At the end of the feeding period, larvae were dissected to remove undigested food from the gut. In the case of parasitized larvae, the dissected hosts were examined under a microscope and the parasites were collected from the haemocoel. The carcasses of the individual M. sexta larvae including their emptied guts were dried in an oven at approximately 75C for 72 h and weighed. The parasites from parasitized larvae were counted and those collected from each host larvae were dried together as above. The Wnal dry mass was used as the measure of growth. For statistical purposes, the initial dry mass of the experimental larvae was estimated after determining the average dry/wet mass ratio of 10 larvae sampled from the initial insect population. These larvae were dried as above to determine their water content. The average dry/wet mass ratio was calculated and applied to the wet mass of larvae used in the experiments. The diet remaining at the end of the feeding trials was dried as above and the total consumption calculated as the diVerences in dry mass of the remaining diet plus that of the gut contents and the dry mass of the total diet given to larvae calculated from the known dry mass content of the fresh diet (Thompson et al. 2005a). Protein and carbohydrate intake were then calculated from the known casein and sucrose content of the diet consumed. Fat and nitrogen analyses We estimated the contributions of protein and carbohydrate to larval growth by analyzing the larvae for nitrogen and fat content. The dried carcasses of M. sexta larvae and the combined parasites collected from individual parasitized hosts were powdered with a mortar and pestle. Each sample was divided approximately in half. Fat was extracted from

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one portion and nitrogen analyzed in the second. Fat was extracted by Wltering each sample three times with approximately 1015 volumes of petroleum ether (3560C). The solvent was evaporated from the combined Wltrates and the fat determined gravimetrically with a microbalance. Nitrogen was determined with a Thermo-Finnigan nitrogen/carbon analyzer (Model: Flash EA1112) in accordance with the method outlined in the operating manual (Chapter 8: Analytical Methods, Analytical method for the NC-soils, NC-sediments and NC-Wlters conWguration, pp 184186). Geometric representation and analysis of data The geometric analysis of Raubenheimer and Simpson (1997, 1999) was employed to examine the eVects of dietary macronutrient ratio on growth, nutrient consumption and nutrient utilization by M. sexta larvae. In this analytical approach, protein and carbohydrate consumption are shown as the two axes of a single bivariate plot, with consumption of the two nutrients indicated by points along linear trajectories or nutrient rails deWned by the nutrient ratios of the individual diets. We measured diVerences in feeding response between individual diets by determining the length or distance along the nutrient rails to the points of nutrient intake at 2 and 3 days and until the termination of feeding at the wandering stage for normal larvae and at 2, 3 and 5 days and until the end of feeding by parasitized larvae. Nutrient rail distance was calculated according to Raubenheimer and Simpson (2003) using the Pythagorean theorem where rail distance is the hypotenuse of a right triangle formed by carbohydrate consumption and protein consumption in the bivariate plot. The response of larvae to macronutrient imbalance, that is, the feeding response of larvae to dietary nutrient ratios that deviate from the intake target ratio is further suggested by the relationship between the points of nutrient intake across the nutrient rails for the diVerent diets. The shape of the line connecting the intake points, referred to as a nutrient intake array, varies depending upon the larval response to excesses and deWciencies in macronutrients (Raubenheimer and Simpson 2003). The intake target ratio for Wfth instar M. sexta larvae, demonstrated experimentally, is approximately 1:1 protein to carbohydrate, (Thompson and Redak 2005). Statistics The eVects of dietary macronutrient ratio and parasitism on growth and nutrient intake by M. sexta were principally analyzed by analysis of variance (ANOVA) and analysis of covariance (ANCOVA) using initial mass as the primary covariate to account for diVerences in initial mass between the experimental host larvae (Raubenheimer and Simpson

1992; Horton and Redak 1993). Analyses were also conducted using additional covariates and multiple covariates as indicated. Unless otherwise indicated, ANCOVA data are presented as least square means SE (standard error). Normality and homogeneity of variance were established with the ShapiroWilk W test and normal probability plots. Preliminary analyses determined that the data met the assumptions of analysis of variance. Accelerated failure time analysis was employed to examine the eVects of diet on the end time for feeding by normal and parasitized larvae (Fox 2001; Lee et al. 2002). All statistics were performed using SAS (version 9.02, 2003; SAS Institute Inc., Cary, NC, USA).

Results EVects of dietary nutrient ratio and parasitism by C. congregate on nutrient consumption and larval development time of M. sexta Cumulative consumption of protein and carbohydrate by M. sexta larvae on the various diets is illustrated as a standard bivariate plot in Fig. 1. For normal and parasitized larvae, nutrient rail distance, the distance along the nutrient rail from the origin to the point of nutrient intake, was aVected by dietary nutrient ratio throughout the feeding period (Table 1). The feeding response of larvae to excesses in carbohydrate and protein were not equal. Analysis of nutrient rail distance demonstrated that larvae generally consume much more on diets having excess carbohydrate with nutrient ratios less than 1, than was the case with diets having nutrient ratios greater than 1 and equivalent excesses of protein (Fig. 2). This was most apparent with normal larvae feeding to the end, where the actual times of feeding were longer on the most carbohydrate-biased diet. Failure time analysis established a signiWcant eVect of parasitism on overall feeding time (X2 = 580.10, df = 1, P < 0.0001). Parasitized larvae fed for signiWcantly longer times than normal larvae (see Fig. 1). Dietary nutrient ratio had a signiWcant eVect on the time to the wandering stage by normal larvae (X2 = 372.71, df = 8, P < 0.0001), with larvae on the C20:100S diet feeding for the longest duration. The diet, however, did not aVect the time to end of feeding by parasitized insects (X2 = 4.99, df = 8, P = 0.7582). EVects of dietary nutrient ratio and parasitism by C. congregata on growth of M. sexta The Wnal mass of M. sexta larvae was signiWcantly aVected by dietary nutrient ratio (F = 6.27, P < 0.0001) and parasitism (F = 584.45, P < 0.0001). There was no interaction

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J Comp Physiol B (2008) 178:515527 Fig. 1 Bivariate plot of protein and carbohydrate consumption by Wfth instar M. sexta larvae (a) and larvae parasitized by C. congregata (b) fed a chemically deWned artiWcial diet having nine diVerent macronutrient ratios. Lines show nutrient rails representing the trajectory of nutrient intake expected for larvae feeding on the individual diets. Nutrient intake points indicate actual nutrient intake at 2 and 3 days, and at the wandering stage for normal larvae, and 2, 3 and 5 days and end of feeding for parasitized larvae (data are raw means). Dietary nutrient ratios are shown relative to the level of each nutrient: P, protein (casein); C, carbohydrate (sucrose) in g l1. Mean times to reach wandering or for the end of feeding are in parentheses at the ends of the nutrient rails

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Table 1 ANCOVA summary demonstrating the eVects of dietary nutrient ratio on nutrient rail distance and nutrient intake arrays of normal Wfth instar M. sexta larvae and larvae parasitized by C. congregata maintained on a chemically deWned artiWcial diet (see Figs. 1, 2)

df Normal (non-parasitized) 2 day Dietary nutrient ratio Initial mass (covariate) 3 day Dietary nutrient ratio Initial mass (covariate) End of feeding (wandering) Dietary nutrient ratio Initial mass (covariate) Parasitized 2 day Dietary nutrient ratio Initial mass (covariate) 3 day Dietary nutrient ratio Initial mass (covariate) 5 day Dietary nutrient ratio Initial mass (covariate) End of feeding Dietary nutrient ratio Initial mass (covariate) 8 1 8 1 8 1 8 1 8 1 8 1 8 1

Mean square

F value

Probability

0.0972 0.4568 0.3366 1.0299 5.3537 0.0487

11.08 52.08 14.51 44.14 103.29 0.94

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.3351

0.0279 0.0139 0.0681 0.0462 0.1343 0.0710 0.2701 0.0143

2.91 1.45 3.53 2.38 3.89 2.05 2.07 0.11

0.0089 0.2345 0.0023 0.1275 0.0011 0.1574 0.0543 0.7420

between dietary nutrient ratio and parasitism; diet inXuences normal and parasitized larvae in a similar manner. These results conWrm those reported previously (Thompson

et al. 2005a; Thompson and Redak 2005) demonstrating that non-parasitized larvae grow best on diets having nutrient ratios close to the intake target ratio of 1:1 (Thompson

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520 Fig. 2 Nutrient rail distances for Wfth instar M. sexta larvae (a) and larvae parasitized by C. congregata (b) feeding for various times on a chemically deWned artiWcial diet having nine diVerent macronutrient ratios. Data are least square means (SE). The scaling for nutrient rail distance is not equivalent in (a) and (b). Nutrient rail distance was calculated according to Raubenheimer and Simpson (2003) using the Pythagorean theorem where rail distance is the hypotenuse of a right triangle formed by carbohydrate consumption and protein consumption in the bivariate plot. Values followed by the same letter are not statistically diVerent (P < 0.05) between diets. Dietary nutrient ratios are shown relative to the level of each nutrient: P, protein (casein); C, carbohydrate (sucrose) in g l1 (see Table 1)

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and Redak 2005), with progressively lower growth as diets become carbohydrate-biased or protein-biased (data not shown). On all diets, growth of parasitized larvae was less

than that of normal larvae (data not shown). Because we were ultimately interested in whether dietary nutrient ratio and parasitism inXuence nutrient utilization, we determined

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the eVects of dietary nutrient ratio and parasitism on growth independent of diVerences in nutrient consumption through a multi-covariate ANCOVA employing protein and carbohydrate consumption as joint covariates. After accounting for diVerences in nutrient consumption, this analysis demonstrated that dietary nutrient ratio and parasitism each aVect the Wnal mass. In this analysis, there was a strong interaction between dietary nutrient ratio and parasitism, indicating that dietary nutrient ratio diVerentially aVects normal and parasitized larvae (Table 2). The mean Wnal mass of normal larvae was similar among diets having intermediate nutrient ratios, but was lower on diets with more extreme nutrient ratios, particularly in the case of protein-biased diets (Fig. 3). Parasitized larvae appeared to
Table 2 ANCOVA summary demonstrating the eVects of dietary nutrient ratio and parasitism by C. congregata on growth (Wnal mass) of M. sexta larvae reared on a chemically deWned artiWcial diet employing carbohydrate and protein consumption as joint covariates (see Fig. 3) df Mean square F value Probability

exhibit a similar pattern of growth with diet, but in no case was there a signiWcant diVerence between larvae on diVerent diets (Fig. 3). With the exception of diets having extreme dietary nutrient ratios, parasitized larvae displayed signiWcantly lower growth than normal larvae. There were also signiWcant eVects of both nutrient covariates, substantiating the importance of protein and carbohydrate consumption for growth (Table 2). The inXuence of protein on Wnal mass was much greater than that of carbohydrate. EVects of dietary nutrient ratio and parasitism by C. congregate on nitrogen and lipid content of M. sexta The results of MANCOVA for nitrogen and lipid content of normal and parasitized M. sexta larvae demonstrated highly signiWcant eVects of parasitism and dietary nutrient ratio (Table 3). No interaction between parasitism and dietary nutrient ratio was evident, indicating that dietary nutrient ratio aVects lipid and nitrogen content similarly in normal and parasitized insects. The larval nitrogen content was similar for all animals fed on diets having nutrient ratios greater than 1:1 (Fig. 4), suggesting that these insects regulate their nitrogen content. Insects on diets having ratios less than 1:1 exhibited lower nitrogen content. Lipid content generally increased as the proportion of dietary carbohydrate increased. Parasitized larvae, being smaller, contained lesser amounts of protein and lipid than normal larvae (Fig. 4). EVects of dietary nutrient ratio and parasitism by C. congregata on utilization of dietary protein and carbohydrate by M. sexta We examined the eVects of dietary nutrient ratio and parasitism on the eYciency of conversion of dietary nitrogen in M. sexta by conducting an ANCOVA on host larval nitrogen content utilizing nitrogen consumption as the covariate. Dietary nutrient ratio, parasitism, and the nitrogen consumption covariate each aVected the nitrogen content of the larvae (Table 4). There was an interaction between dietary nutrient ratio and parasitism, but not between these eVects and nitrogen consumption. Thus, within the range of nitrogen consumed for each diet, the eVect of nitrogen consumption on larval nitrogen content was similar. For all diets, nitrogen content was positively related with nitrogen consumption (slope = 428.11 mg g1 25.76 SE). Covariate adjusted nitrogen values showing the interaction of dietary nutrient ratio and parasitism are illustrated in Fig. 5a. Larger values represent net higher nitrogen conversion. Conversion eYciency was greatest on diets having intermediate dietary nutrient ratios and less on the extreme carbohydrate-biased or protein-biased diets. In general, these Wndings are consistent with the results on growth after

Final mass Dietary nutrient ratio (DNR) Parasitism (P) DNR P interaction Covariates Protein consumption Carbohydrate consumption Initial mass 1 1 1 0.6228 0.0774 0.0297 76.38 9.49 3.64 <0.0001 0.0025 0.0584 8 1 8 0.1879 0.6110 0.3103 23.05 74.93 3.81 <0.0001 <0.0001 0.0005

Fig. 3 Growth of Wfth instar M. sexta larvae (open bars) and larvae parasitized by C. congregata (dark bars) fed on a chemically deWned artiWcial diet having nine diVerent macronutrient ratios. Data are least square means (SE). Values followed by the same letter (upper case for normal, lower case for parasitized) are not statistically diVerent (P < 0.05) between diets. SigniWcant diVerences between normal and parasitized larvae on individual diets are shown by an asterisk. Dietary nutrient ratios are shown relative to the level of each nutrient: P, protein (casein); C, carbohydrate (sucrose) in g l1 (see Table 2)

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522 Table 3 MANCOVA summary demonstrating the eVects of dietary nutrient ratio and parasitism by C. congregata on nitrogen and lipid content in Manduca sexta larvae reared on a chemically deWned artiWcial diet (see Fig. 4)

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Characteristic root

Percentage of variance explained

Pillais trace

F value (df)

Probability

Treatment Dietary nutrient ratio (DNR) Parasitism (P) DNR P interaction Initial mass (covariate) 1.1620 2.4639 0.0967 0.0464 84.79 100.00 78.54 100.00 0.7010 0.7113 0.1139 0.0440 8.39(16,244) 149.07(2,121) 0.92(16,244) 2.79(2,121) <0.0001 <0.0001 0.5454 0.0657

Table 4 ANCOVA summary demonstrating the eVects of dietary nutrient ratio and parasitism by C. congregata on nitrogen and lipid content of M. sexta larvae reared on a chemically deWned artiWcial diet employing nitrogen and carbohydrate consumption as covariate, respectively df Nitrogen content Dietary nutrient ratio (DNR) Parasitism (P) DNR P interaction Covariate Nitrogen consumption Lipid content Dietary nutrient ratio (DNR) Parasitism (P) DNR P interaction Covariate Carbohydrate consumption Fig. 4 Bivariate plot of nitrogen and lipid content of Wfth instar M. sexta larvae (dark symbols) and larvae parasitized by C. congregata (light-coloured symbols) fed on a chemically deWned artiWcial diet having nine diVerent macronutrient ratios. Diets with P:C ratios > 1 are shown with closed symbols; diets with P:C ratios < 1 are shown with open symbols. Squares indicate P:C ratio = 1, the approximate intake target ratio. Data are least square means (SE). Dietary nutrient ratios are shown relative to the level of each nutrient: P, protein (casein); C, carbohydrate (sucrose) in g l1 (see Table 3) 1 17572.4823 16.86 <0.0001 8 1 8 2695.9499 18284.1102 1391.1586 2.56 17.54 1.33 0.0119 <0.0001 0.2323 1 16992.1417 276.03 <0.0001 8 1 8 1412.9301 477.9131 377.0303 22.95 7.76 6.12 <0.0001 0.0061 <0.0001 Mean square F value Probability

EVect of initial mass was insigniWcant (see Fig. 5)

considering diVerences in nutrient intake between diets described above. The mean nitrogen content of parasitized larvae was generally lower than that of normal larvae on protein-biased diets, and similar to normal larvae on carbohydrate-biased diets. The overall diVerence among diets was signiWcant, but small, with normal larvae having a nitrogen content of 96.56 mg larva1 1.13 SE, and parasitized larvae 90.12 mg larva1 1.53 SE (t = 2.79, P = 0.0061), an approximate 7% diVerence. Results on the eYciency of conversion of carbohydrate to lipid demonstrate that the covariate carbohydrate consumption, dietary nutrient ratio and parasitism, all inXuenced lipid content (Table 4). However, there were no

interactions between any of the treatments, including the covariate, carbohydrate consumption. Lipid content generally increased with carbohydrate consumption (slope = 55.05 mg g1 13.41 SE) and with decreased dietary carbohydrate after considering diVerences in consumption (Fig. 5b). For all diets, however, parasitized insects had signiWcantly lower mean lipid levels than normal larvae: 70.28 mg parasitized larva1 7.41 SE, 117.55 mg normal larva1 5.15 SE (t = 4.19, P < 0.0001), an approximate 40% diVerence. EVects of dietary nutrient ratio and host nutrient consumption on C. congregata Parasite numbers and total parasite biomass developing in individual host larvae were highly variable in the present experiments, and ANOVA failed to demonstrate any eVect of dietary nutrient ratio (F(8,56) = 1.26, P = 0.2835; F(8,56) = 1.33, P = 0.2492, respectively). In contrast, our earlier investigation

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Fig. 5 Nitrogen and lipid content of Wfth instar M. sexta larvae and larvae parasitized by C. congregata fed on a chemically deWned artiWcial diet having nine diVerent macronutrient ratios. a Interaction plot of nitrogen content for normal (closed bars) and parasitized larvae (open bars). Values followed by the same letter (upper case for normal, lower case for parasitized) are not statistically diVerent (P < 0.05) between diets. SigniWcant diVerences between normal and parasitized larvae on

individual diets are shown with an asterisk. b Lipid content for dietary nutrient ratios averaged across levels of parasitism. Values followed by the same letter are not statistically diVerent (P < 0.05) between diets. All data are least square means (SE). Dietary nutrient ratios are shown relative to the level of each nutrient: P, protein (casein); C, carbohydrate (sucrose) in g l1 (see Table 4)

demonstrated a correlation between dietary nutrient ratio with both parasite numbers and biomass (Thompson et al. 2005a). We believe the diVerence in results from these studies is due, in part, to the deposition of more variable parasite egg numbers during the present experiments and reXects the diYculty in attaining uniform oviposition by female wasps, as discussed earlier (Thompson et al. 2005b). There was, however, a signiWcant eVect of dietary nutrient ratio on average parasite biomass (F(8,56) = 3.15, P = 0.0051). Average parasite mass was greater on the 90C:30S diet, but not diVerent between any of the other diets (data not shown). Thompson et al. 2005b demonstrated the dependence of parasite development on blood protein, amino acid and trehalose concentrations; the latter, in turn, was inXuenced by host protein and carbohydrate consumption. During the present study, therefore, we conducted an ANCOVA, using host protein and carbohydrate consumption as joint covariates, to determine the eVects of dietary nutrient ratio on parasite development after accounting for diVerences in host nutrient consumption. Dietary nutrient ratio and the covariate protein consumption inXuenced both parasite numbers and total parasite biomass developing in individual host larvae (Table 5). There was no interaction between dietary nutrient ratio and protein consumption. Host protein consumption, therefore, inXuenced parasites in a similar manner for all diets. Carbohydrate consumption did not have a signiWcant eVect on parasite number or total parasite biomass.

Table 5 ANCOVA summary demonstrating the eVects of dietary nutrient ratio on the number and total biomass of C. congregata developing in M. sexta larvae employing host nutrient and carbohydrate consumption as joint covariates df Mean square F value Probability Parasite number Dietary nutrient ratio Covariate Protein consumption Total parasite biomass Dietary nutrient ratio Covariate Protein consumption Average parasite biomass Dietary nutrient ratio Covariate Protein consumption 1 0.0947 0.0216 2.17 0.49 0.1467 0.4850 Carbohydrate consumption 1 8 0.1192 2.73 0.0133 1 24897.4745 281.4802 8.91 0.10 0.0043 0.7522 Carbohydrate consumption 1 8 4046.6383 1.45 0.1985 1 12646.8142 13630.7101 16.02 1.72 0.0002 0.1947 Carbohydrate consumption 1 8 13490.8095 1.71 0.1180

Host carbohydrate and protein consumption did not aVect the average parasite biomass after accounting for diVerences in nutrient consumption between diets (Table 5). Dietary nutrient ratio did aVect the average biomass, with parasites developing in hosts on the 90C:30S diet having the highest average mass. The result is unex-

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plained, and if this group were excluded from the analysis there would be no eVect of diet. Thus, the results were generally similar to those reported previously where mean average biomass was unaVected by dietary nutrient ratio (Thompson et al. 2005b). EVects of dietary nutrient ratio on nitrogen and lipid content of C. congregata The relationship between the nitrogen and lipid content of individual parasite larvae is shown in Fig. 6. MANOVA demonstrated a signiWcant eVect of dietary nutrient ratio (Pillais Trace F(16,88) = 2.43; P = 0.0043), but with the exception of parasites from hosts on the 90C:30S diet, where parasites were unusually large, the lipid and nitrogen content of parasites was similar on all diets. Indeed, when the 90C:30S group was excluded, the MANOVA failed to demonstrate any eVect of diet (Pillais Trace F(14,80) = 1.15; P = 0.3298) and the overall levels of nitrogen and lipid in individual parasites were 37 3 and 53 3 g (x SE), respectively. Developing parasites, therefore, do not appear to regulate their nitrogen content as did the host M. sexta. EVects of dietary nutrient ratio on utilization of host dietary protein and carbohydrate by C. congregata We also examined the eVects of dietary nutrient ratio on the eYciency of host nutrient conversion by C. congregata. ANCOVA employing host nitrogen consumption as the

covariate demonstrated that dietary nutrient ratio inXuenced nitrogen content of parasites (F = 4.61; P < 0.0001), but there was no eVect of nitrogen consumption (F = 0.01, P = 0.9420). There was an interaction between dietary nutrient ratio and the nitrogen consumption, but this was entirely due to the results for parasites from hosts on the 90C:30S diet. The interaction was absent when this group was excluded. Regarding the conversion of carbohydrate to lipid, neither dietary nutrient ratio nor the covariate carbohydrate consumption aVected the parasite lipid content (F = 1.11; P = 0.3708; F = 0.23, 0.6323, respectively).

Discussion The results of the present study considerably expand on those of our previous investigations to demonstrate the impact of host nutrition on the development of an insect parasitoid. Parasitic associations of insect parasitoids are ultimately virulent and host death is imminent (Ewald 1995). Once parasitized, the host plays no role in the evolution of the parasitoidhost relationship. Nevertheless, during the development of most endoparasitoids, the host lives, continuing to grow and feed for extended periods, often longer than the duration of normal larval development, during which time the parasite grows and develops (Quickie 1997). Here, we demonstrate for the Wrst time how parasitism by the gregarious species C. congregata aVects the overall host nutrient consumption and utilization by M. sexta, and how the host partitions nutrients between itself and the developing parasites over the entire Wfth stadium of development. M. sexta larvae respond to dietary nutrient imbalance by altering diet consumption. Being unable to reach their intake target ratio while feeding on individual unbalanced diets, the larvae adjusted their intake to compensate for excesses and deWciencies in macronutrients. The normal and parasitized larvae did so in diVerent ways. The nutrient intake arrays for normal larvae form arcs throughout the Wfth stadium, and these are particularly displaced at wandering on diets having greater proportions of carbohydrate. Although the nutrient intake arrays for parasitized larvae are not precisely linear, they are suggestive of the equal distance rule (Simpson and Raubenheimer 1993a, b) or Wxed proportion regulatory pattern (Raubenheimer and Simpson 2003) observed in other generalist feeders including the grasshopper, Schistocerca gregaria (Simpson and Raubenheimer 1993a, b; 1997) and the caterpillar Spodoptera littoralis (Lee et al. 2002), where insects consume excesses of one nutrient, protein or carbohydrate, in order to obtain adequate amounts of the other. In those cases, the responses were near equivalent for both nutrients. With M. sexta, the response was not equivalent for both nutrients.

Fig. 6 Bivariate plot of nitrogen and lipid content of C. congregata larvae developing in Wfth instar M. sexta larvae fed on a chemically deWned artiWcial diet having nine diVerent macronutrient ratios. Data are means (SE). Dietary nutrient ratios are shown relative to the level of each nutrient: P, protein (casein); C, carbohydrate (sucrose) in g l1

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As reXected by increased nutrient rail distance, normal larvae on diets having excess carbohydrate, dietary nutrient ratios less than 1:1, consumed much more diet than was the case with diets having equivalent excesses of protein. This demonstrates that larvae respond more strongly to deWciencies in protein, than in carbohydrate, and suggests that larvae may have greater diYculty processing excess protein than excess carbohydrate. For normal larvae, this was also reXected by diVerences in the Wnal mass between diets, where larvae clearly grew best on diets having dietary nutrient ratios close to 1:1, the intake target ratio, with the lowest mean growth on the most protein-biased diet. The nutrient rail distances for parasitized larvae were more uniform between diets, but there still was a slight bias towards increased consumption on diets having higher carbohydrate. The Wnding that growth of parasitized larvae, although lower than that of non-parasitized insects, was uniform with variations in dietary nutrient ratio, consistent with the results for nutrient consumption and with the results of our earlier investigation demonstrating that parasitized larvae fail to regulate macronutrient intake and fail to display a speciWc intake target ratio when oVered dietary choices (Thompson and Redak 2005). We suggest that absence of a speciWc intake target accompanied by decreased and uniform growth allows parasitized larvae to accept a broad range of nutritional conditions that would be unacceptable to normal larvae. This, in turn, may enable parasitized insects to feed on areas within plants that are less nutritionally suitable and not preferred by normal larvae, thereby decreasing competition and providing an adaptive advantage for survival of parasitized larvae and successful development of parasites. This hypothesis is yet to be tested. The altered feeding response and reduced growth of parasitized larvae fulWll a number of the criteria outlined by Poulin (1995) for being adaptive. They are complex, show signs of purposeful design and have arisen in several lineages of hosts and insect parasites. The alternate hypothesis that reduced growth is merely a pathogenic response to parasitism, however cannot be excluded until a Wtness beneWt for the parasite can be demonstrated. Despite the responses of normal and parasitized M. sexta to dietary nutrient ratio, our results clearly indicate that larvae display post-ingestive regulation of protein utilization. On diets having dietary P:C ratios greater than 1:1 (proteinbiased diets), larvae display high conversion eYciencies and contain a similar amount of nitrogen regardless of the amount of nitrogen ingested. In this regard, the results of M. sexta are similar to those reported for larvae of S. littoralis (Lee et al. 2002). Further, our results are consistent with the eVects of dietary nutrient ratio on nutrient consumption, where larvae displayed diVerential responses to diets having excess protein or excess carbohydrate as dis-

cussed above. Excess nitrogen is likely eliminated through increased uric acid production as suggested for other species (Lee et al. 2002; Zanatto et al. 1993). Also, when carbohydrate intake is very low, the deaminated protein substrate also contributes to net carbohydrate synthesis and the formation of trehalose through gluconeogenesis (Thompson et al. 2003). Unlike the case of S. litoralis (Lee et al. 2002), we did not observe a dramatic reduction of nitrogen conversion eYciency on the extreme proteinbiased diet; S. littoralis, however, displayed higher protein consumption on protein-biased diets than did M. sexta. Despite increased consumption on carbohydrate-biased diets, M. sexta larvae generally have lower protein content as the dietary proportion of protein decreases. Lipid content of both normal and parasitized larvae generally increased as the proportion of dietary carbohydrate increased. An important Wnding in these investigations was that after accounting for diVerences in carbohydrate consumption among diets, the lipid content of parasitized larvae was dramatically less than that of normal larvae. Although nitrogen content of parasitized larvae was also less after diVerences in nitrogen consumption between diets was considered, the diVerence was slight. We suggest that the low level of host lipid accumulation represents the principal energetic cost of supporting parasite development. Although previous investigations suggest that one mechanism enabling insects to eliminate excess carbohydrate is through increased respiration (Zanotto et al. 1993, 1997; Lee et al. 2002), a diVerence in respiration between parasitized and normal larvae is unlikely to contribute to the variation observed in lipid levels. Studies by others failed to demonstrate any eVect of parasitism on CO2 production by M. sexta during the feeding phase of the Wfth stadium (Alleyne et al. 1997). Lipid synthesis and storage in insects occur principally in the perivisceral fat body, an organ surrounding that gut and responsible for regulating much of intermediary metabolism (Keeley 1985; Haunerland and Shirk 1995). Previous studies suggest that parasitism signiWcantly aVects fat body diVerentiation and function in M. sexta larvae. Dahlman and Green (1981) reported that perivisceral fat body is substantially diminished in parasitized larvae, a Wnding supported by our own observations (unpublished), and consistent with decreased lipid accumulation. Metabolic studies with parasitized M. sexta demonstrate a shift of dietary carbohydrate away from lipid synthesis into trehalose, sometimes accompanied by an elevation in blood trehalose level (Thompson 1993). Further, there occurs a simultaneous increase in gluconeogenesis, presumably in the peripheral subcutaneous fat body, which is induced in parasitized larvae under nutritional conditions that fail to stimulate de novo carbohydrate synthesis in normal larvae (Thompson 2001; Thompson et al. 2002). Considering

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these metabolic alterations, it is not surprising that dietary carbohydrate consumption is of lesser importance than protein consumption. Indeed, the present results conWrm those of our previous investigations indicating that host protein consumption is the principal factor supporting successful parasite development. In contrast to the results with host larvae, C. congregata do not appear to regulate either their nitrogen or lipid content. The larvae had similar nitrogen and lipid levels regardless of the host diet. This undoubtedly reXects the generally uniform size of both the parasitized hosts and individual parasites, and the fact that host blood protein nitrogen and trehalose levels that serve to nourish parasites are maintained within a relatively narrow range over the varied nutritional conditions employed in the present investigation (Thompson et al. 2005a, b). The depressed growth of parasitized hosts and their resultant uniform size likely have physiological as well as ecological relevance for successful parasite development. In conclusion, M. sexta larvae respond to variations in dietary nutrient ratio and nutrient imbalances by altering their diet consumption, reXecting potential trade-oVs between growth and the costs of the physiological responses to excesses and deWciencies in macronutrients. Regulation of feeding behaviour in response to dietary nutrient composition is complex, involving modulation of peripheral chemoreception, nutrient-allelochemical interactions, feedback through blood chemistry, as well as feeding experience and learning (Simpson and White 1990; Simpson and Raubenheimer 1993a, b; 1996; Glendinning and Slansky 1994; Thompson et al. 2001; Thompson and Redak 2007). How these mechanisms are integrated with nutrient availability and the insects nutritional state is poorly understood. Recently, authors have speculated about the involvement of biogenic amines, neuropeptides and other hormones in feeding regulation (Rogers et al. 2004; Cohen et al. 1988, 2002; Thompson et al. 2001; Bede et al. 2007). The present study demonstrates that parasitism by C. congregata perturbs the mechanisms that regulate feeding by M. sexta, and, moreover, appears to do so in a manner that contributes to the nourishment of the parasites. Studies on parasitized insects provide a unique opportunity to examine the essential roles of various mechanisms in eVecting feeding. Previous investigations on parasitized caterpillars demonstrate changes in responsiveness to allelochemicals and in nutrient-allelochemical interactions (Slansky 1992; Singer et al. 2004; Thompson and Redak 2007). Changes in hormone titers are also well documented in parasitized insects (Adamo and Shoemaker 2000; Beckage and Gelman 2001). Future investigations will attempt to establish physiological correlates more directly with feeding behaviour and nutrient intake.

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