Ecology Letters, (2011) 14: 453–461 doi: 10.1111/j.1461-0248.2011.01604.x LETTER indirectly increase virulence and transmission potential of a monarch butterfly parasite by reducing defensive chemistry of a shared food plant

Abstract Jacobus C. de Roode1*, Rachel M. Parasites and hosts live in communities consisting of many interacting species, but few studies have examined Rarick1, Andrew J. Mongue1, Nicole how communities affect parasite virulence and transmission. We studied a food web consisting of two species M. Gerardo1 and Mark D. Hunter2 of milkweed, two milkweed herbivores (monarch butterfly and oleander ) and a monarch butterfly- 1Department of Biology, Emory specific parasite. We found that the presence of aphids increased the virulence and transmission potential of the University, 1510 Clifton Road, monarch butterflyÕs parasite on one milkweed species. These increases were associated with aphid-induced Atlanta, GA 30322, USA decreases in the defensive chemicals of milkweed plants. Our experiment suggests that aphids can indirectly 2Department of Ecology and increase the virulence and transmission potential of monarch butterfly parasites, probably by altering the Evolutionary Biology, University of chemical composition of a shared food plant. These results indicate that species that are far removed from Michigan, 1141 Natural Sciences host–parasite interactions can alter such interactions through cascading indirect effects in the food web. Building, 830 North University, As such, indirect effects within ecological communities may drive the dynamics and evolution of parasites. Ann Arbor, MI 48109–1048, USA *Correspondence: E-mail: Keywords [email protected] Aphis nerii, , cardenolide, Danaus plexippus, disease ecology, host–parasite interaction, Ophryocystis elektroscirrha, trait-mediated, tri-trophic, virulence evolution.

Ecology Letters (2011) 14: 453–461

result in increased absolute or relative abundance of host species, INTRODUCTION resulting in greater disease prevalence and severity (e.g. Mitchell et al. Host–parasite models have shown that host density as well as parasite 2002; Keesing et al. 2006; Johnson et al. 2008; Borer et al. 2009). Many virulence (i.e. parasite-induced host mortality) and transmission play of these studies have dealt with generalist parasites, where the relative key roles in the spread of parasites in host populations (Anderson & abundance of more and less competent host species will affect the May 1991; Hudson et al. 2002). A major limitation of these models is overall disease prevalence and severity in a population. However, that they generally represent host and parasite species in isolation, and studies on the effects of community structure on host-specific assume that transmission and virulence are under the full control of parasites are still rare. Moreover, few studies have investigated the role host and parasite (reviewed in Restif & Koella 2003). However, hosts of trait-mediated indirect effects (Raffel et al. 2008, 2010) on parasite and parasites are members of larger communities of interacting virulence and transmission, two traits that crucially affect the spread of species (Lafferty et al. 2006), which may impact host density, parasite parasites in populations and the severity of disease outbreaks transmission and virulence, and thereby influence parasite dynamics (Anderson & May 1991; Hudson et al. 2002). and disease outbreaks. Here, we studied how community composition may affect the Studies in community ecology have long shown that species can virulence and transmission of a host-specific parasite through indirect affect each other directly and indirectly. Indirect effects occur when effects. We used a naturally occurring food web consisting of four one species affects the performance of a second species through its species and three trophic levels: milkweed plants ( effect on a third. They can not only occur due to changes in species or Asclepias incarnata), two milkweed herbivores (the monarch butterfly density (density-mediated indirect effects), but may also be mediated Danaus plexippus and the oleander aphid Aphis nerii) and a monarch by traits that are involved in the interaction between species (trait- butterfly-specific protozoan parasite (Ophryocystis elektroscirrha) (Fig. 1). mediated indirect effects: Wootton 1994; Abrams 1995; Werner & This food web provides a suitable system to study the indirect effects Peacor 2003). For example, one herbivore may induce a phenotypic on virulence and transmission for several reasons. increase in defence traits in its food plant, which may subsequently First, virulence and transmission potential of the parasite affect a second herbivore species (Van Zandt & Agrawal 2004). O. elektroscirrha (McLaughlin & Myers 1970) are relatively straightfor- Like predators and competitors, parasites are well known to affect ward to measure. This parasite forms dormant spores on the outside the interactions between other species (MacNeil et al. 2003; Packer of adult monarch butterflies. Spores are transmitted to eggs and et al. 2003; Mouritsen & Poulin 2005; Hatcher et al. 2006). There is milkweed during oviposition, after which hatching larvae become also increasing evidence that different parasite species can affect each infected by ingesting them. The parasite then replicates during larval otherÕs virulence and transmission when co-infecting the same host and pupal stages and forms spores on the newly developed adult (Graham 2008; Jolles et al. 2008; Telfer et al. 2010). However, little is butterfly. Spores replicate no further on the adult butterfly, and higher known about the indirect effects of other free-living species on host– sporeloads result in greater transmission to larvae (De Roode et al. parasite interactions. A number of studies have addressed density- 2008b, 2009). Thus, the sporeload on an adult butterfly provides a mediated indirect effects, and shown that lower species diversity can measure of the full transmission potential of the parasite. Besides

2011 Blackwell Publishing Ltd/CNRS 454 J. C. de Roode et al. Letter

Figure 1 A food web consisting of milkweed (Asclepias curassavica or Asclepias incarnata), aphids (Aphis nerii), monarch butterfly caterpillars (Danaus plexippus) and a parasite of the monarch butterfly (Ophryocystis elektroscirrha). Solid arrows show the direction of energy flow. The dashed arrows depict the indirect effects of aphids on the interaction between the monarch butterfly and its parasite. Results suggest that aphids have a negative effect on the defensive chemistry of A. curassavica (indicated with the Ô)Õ sign), which subsequently increases the virulence and transmission potential of the monarch butterflyÕs parasite (indicated with a Ô+Õ sign). However, as aphids do not alter the chemistry of A. incarnata, aphids are not expected to indirectly affect the interaction between the monarch butterfly and its parasite when monarchs feed on this species (hence the absence of dashed lines connecting aphids with A. incarnata and the interaction between the monarch butterfly and its parasite).

providing greater transmission, higher sporeloads also result in greater tested this hypothesis by rearing monarch caterpillars on A. curassavica virulence, with heavily infected monarchs having reduced eclosion and (high cardenolide) and A. incarnata (low cardenolide) plants with or mating success and reduced adult lifespan (De Roode et al. 2008b). without aphids, and by measuring parasite virulence, parasite Second, monarch butterfly caterpillars are specialists on milkweed transmission potential and cardenolide concentrations. We predicted plants and their close relatives (Ackery & Vane-Wright 1984). that aphids would reduce the cardenolide concentrations of the high- Milkweeds are toxic to most herbivores as they produce secondary cardenolide A. curassavica and thereby indirectly increase parasite chemicals called cardenolides (Malcolm & Zalucki 1996; Agrawal & virulence and transmission potential in monarchs reared on this Fishbein 2006). Monarchs suffer little from these chemicals, and in species. In contrast, we predicted no such effects on the low- fact sequester them to protect themselves against predators (Brower & cardenolide species A. incarnata. Fink 1985). Recent studies have also suggested that these chemicals can reduce the virulence and transmission potential of the monarchÕs MATERIALS AND METHODS protozoan parasite O. elektroscirrha (De Roode et al. 2008a; Lefe`vre Monarch, aphid, plant and parasite sources et al. 2010). More specifically, these studies found that parasites produced lower sporeloads and caused lower virulence on monarchs Monarchs used in this experiment were the lab-reared grand-offspring reared on a milkweed species with high cardenolide concentrations of butterflies collected in Miami, FL, in March 2008. Monarchs from (tropical milkweed: A. curassavica) than on a species with low three full-sib family lines were allocated randomly to experimental concentrations (swamp milkweed: A. incarnata). treatments. Aphids were the clonal offspring of a single aphid, also Third, monarch caterpillars regularly share their milkweed food collected in Miami, FL, in March 2008. Aphids had been maintained on plants with co-occurring oleander aphids, Aphis nerii (e.g. Koch et al. milkweed by transferring two adult and four mid-instar aphids to fresh 2005), especially late in the season when aphids colonize many plants plants every 2 weeks; aphids and plants were maintained in a climate- within milkweed patches and reach high numbers on individual plants controlled chamber at 22 C, 50% RH and a 16L : 8D light cycle. (Helms et al. 2004; Helms & Hunter 2005). Like monarch butterflies, The parasite (denoted C1F3-P2-1) was a clone derived from a monarch oleander aphids are specialist herbivores on milkweed and sequester butterfly collected in Miami, FL, in 2004. Milkweed seeds were obtained cardenolides as a defence against predation (Malcolm 1991). Impor- from Butterfly Encounters, CA. Seeds were germinated in seedling mix tantly, studies have suggested that high-cardenolide milkweed species and repotted into Fafard 3B (Conrad Fafard, Inc., Agawam, MA, USA) can reduce their cardenolide concentrations in response to aphid soil 2 weeks post-germination. They were maintained in a climate- herbivory (Martel & Malcolm 2004; Zehnder & Hunter 2007b). It is controlled greenhouse until used in the experiment. thought that such reductions may both save resources and limit aphid population growth, as lower levels of cardenolides should increase the Experimental design susceptibility of aphids to predators (Malcolm & Zalucki 1996; Martel & Malcolm 2004; Zehnder & Hunter 2007b). The experiment was fully factorial, with milkweed species (A. curassavica Overall, the studies described above suggest that aphids are likely to vs. A. incarnata), aphid treatment (with or without aphids) and parasite indirectly increase the virulence and transmission potential of treatment (infected vs. uninfected) as fixed factors. Each milkweed monarch butterfly parasites on high-cardenolide milkweeds. This is species by aphid treatment by parasite treatment combination initially because aphids can reduce the cardenolide concentrations of high- had between 10 and 30 replicate larvae, as shown in Table 1. Few cardenolide milkweeds and because lower cardenolide levels correlate larvae died during the experiment, and final numbers on which with higher parasite virulence and transmission potential. Here, we statistical analyses were based are also shown in Table 1.

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Table 1 Experimental treatments and numbers of replicate larvae used for each treatment combination

Milkweed species Aphid treatment Parasite treatment Initial number Number used for analysis Plants used for cardenolides

Asclepias incarnata With aphids Infected 30 26 15 Asclepias incarnata With aphids Uninfected 10 9 – Asclepias incarnata Without aphids Infected 30 29 15 Asclepias incarnata Without aphids Uninfected 10 9 – Asclepias curassavica With aphids Infected 30 27 15 Asclepias curassavica With aphids Uninfected 10 9 – Asclepias curassavica Without aphids Infected 20 19 15 Asclepias curassavica Without aphids Uninfected 10 10 – Total 150 138 60

Numbers of larvae used for the analysis were lower than initial numbers due to some monarch mortality.

On day 0 of the experiment, we randomly assigned A. curassavica Cardenolide identification and quantification and A. incarnata plants to aphid and parasite treatments and placed them into individual 1.34-L plastic tubes with meshed lids. We added On days 0 and 13, we removed a small leaf from a subset of plants two adult and four mid-instar aphids to plants in the aphid treatments, (see Table 1) for chemical analysis. Cardenolide concentrations were and maintained all plants in a climate-controlled chamber held at 26 C, estimated using established methods (Malcolm & Zalucki 1996; 50% RH and a 16L : 8D light cycle. Aphids were counted on days 3, 6, De Roode et al. 2008a). Leaves were collected in glassine envelopes 10 and 13. On day 13, 2-day-old monarch caterpillars were placed and freeze-dried. They were weighed and pulverized using a bead mill, individually in 10-cm petri dishes that contained a moist filter paper and then extracted in 100% methanol. Extracts were analysed using a leaf disc obtained from the individual plant that was assigned to each reverse phase high-performance liquid chromatography (HPLC). caterpillar. Day 13 was chosen to allow aphid populations to grow to Peaks were detected by diode array at 218 nm, and absorbance typical field densities (Helms et al. 2004). Caterpillars in the infected spectra were recorded from 200 to 300 nm with digitoxin as an treatments received 10 parasite spores on their leaf discs, whereas internal standard. Peaks with symmetrical absorption maxima between control caterpillars did not. Upon complete consumption of their leaf 217 and 222 nm were recorded as cardenolides (Malcolm & Zalucki disc (within 24 h), caterpillars were transferred to their assigned plants 1996). We detected a total of 16 different cardenolide peaks in in 1.34-L plastic tubes. Thus, caterpillars completed their development A. curassavica and four in A. incarnata. Concentrations of each peak on individual live plants, and in the case of aphid treatment, aphids were estimated relative to the internal standard, and total cardenolide remained present on these plants. Plants were watered as needed, and concentration was calculated as the sum of all individual cardenolide extra food was provided to caterpillars that ran out of milkweed before peaks. reaching the pupal stage. In such cases, we took care to provide caterpillars with milkweed of the appropriate species that had or did not Statistical analysis have aphids, depending on the treatment. Seven days after pupation (an average of 2 days before eclosion), We used analyses of variance (ANOVAs) to determine the effects of pupae were transferred to a separate room maintained at the same milkweed species and aphid treatment on monarch adult lifespan conditions as the climate-controlled chamber. This was done to avoid index and parasite sporeload. In these analyses, we first fitted full the potential contamination of the larval rearing chamber with spores models with infection treatment, milkweed species, aphid treatment derived from eclosing infected adult monarchs. Once monarchs and the interaction between these factors. Parasite sporeload was eclosed from their pupal cases, they were transferred to individual log10-transformed to ensure homogeneity of variance and normality of glassine envelopes and held in a chamber at 12 C without food and errors. water. We then recorded their time of death and calculated a measure We also used ANOVAs to determine the effects of day (0 vs. 13) and of monarch adult lifespan – referred to from hereon as lifespan index aphid treatment on overall cardenolide concentrations and the – as the difference (in days) between the day of eclosion and the day concentrations of individual cardenolides in milkweed plants. These of death. Lifespan index provides a combined index of adult monarch analyses were carried out separately for A. curassavica and A. incarnata. lifespan and starvation resistance. We have shown previously that the Where necessary, the concentrations of individual cardenolides were lifespan index responds to parasite infection and increasing parasite square-root-transformed to ensure homogeneity of variance and numbers in a similar way as lifespan under more natural conditions normality of errors. We also used principal components analysis to (De Roode et al. 2009). Although a more natural way of measuring describe the composition of cardenolides in A. curassavica plants (this lifespan might be preferable, allowing monarchs to fly in cages for the was not possible for A. incarnata, as most plants contained only two duration of their life results in the loss of > 90% of parasite spores types of cardenolides). As dictated by the results (below), we analysed (De Roode et al. 2009), resulting in highly inaccurate estimates of certain cardenolide data for day 13 (when caterpillars were infected) parasite transmission potential. separately to confirm that cardenolides differed between plants with After monarchs died, we removed their wings and vortexed their and without aphids on this day. We then used ANOVA to investigate the bodies in 5 mL H2O for 5 min to shake off parasite spores. Spores effect of day and aphid treatment on all principal components that were subsequently counted using a hemocytometer to provide a explained > 10% of the variance of the cardenolide composition. measure of parasite transmission potential (sporeload). We also used linear models to analyse the relationship between

2011 Blackwell Publishing Ltd/CNRS 456 J. C. de Roode et al. Letter cardenolides (overall concentration, pca1 and individual concentra- high-cardenolide A. curassavica and on the low-cardenolide A. incarnata tions) and parasite sporeload; these analyses were restricted to (Fig. 2a, grey bars), and infected adult monarchs had a similar lifespan cardenolides measured on day 13 when plants were used to infect index when reared on either milkweed species (Fig. 2b, grey bars). caterpillars. The analyses were also restricted to cardenolides that These results suggest that the presence of aphids increased parasite differed significantly on day 13 due to the presence of aphids, so as to sporeload and reduced monarch adult lifespan index on A. curassavica reduce the inflation of Type I error rates. Thus, this analysis was carried to similar levels as on A. incarnata (Fig. 2). ANOVAs restricted to out only for cardenolides RT584 and RT650 (see Results section). monarchs reared on A. curassavica indeed confirmed that monarchs All ANOVAs and linear models were carried out in R 2.7.0 reared on A. curassavica carried higher parasite sporeloads (R Foundation for Statistical Computing, Vienna, Austria). Signifi- (F1,44 = 4.46; P = 0.040) and lived shorter lives (F1,44 = 10.8; cance (P < 0.05) was determined by comparing the explanatory power P = 0.002) when reared on plants with aphids than on plants without of models that included or did not include an explanatory variable aphids. using the ANOVA function in R (Crawley 2002). Principal components To confirm that the effect of aphids on monarch lifespan index was analysis was carried out in SAS (SAS Institute Inc., Cary, NC, USA). mediated through effects on parasite sporeload, we included sporeload Aphid population growth was analysed using a linear mixed effects as a covariate in the analysis of monarch lifespan index. This showed model, in which milkweed species and day were fixed effects, and that sporeload was a strong predictor of monarch adult lifespan index milkweed individual was a random effect. The model was fitted using (F1,95 = 76.1, P < 0.0001), but that the interaction between milkweed maximum likelihood. To determine whether milkweed species species and aphid presence (see Fig. 2 legend) was no longer significantly affected aphid reproduction, models with and without significant (F1,95 = 3.15, P = 0.079). These results suggest that aphids the interaction between milkweed species and day were compared can increase the sporeload of O. elektroscirrha on monarchs reared on using the ANOVA procedure in SPLUS 7.0 (Crawley 2002); the degrees of the high-cardenolide A. curassavica and that this increased sporeload freedom and P-value for this comparison are reported. Aphid consequently results in a reduced lifespan index in the monarch numbers were log-transformed prior to the analysis. butterfly host.

RESULTS Effects of aphids on milkweed chemistry Effects of aphid-free milkweed species on virulence and Aphid populations grew rapidly on both milkweed species, but grew transmission of monarch butterfly parasites less rapidly on A. curassavica than on A. incarnata plants (Fig. 4; mixed- effects model: d.f. = 1, P = 0.0008), possibly due to the higher levels As expected on the basis of previous studies (De Roode et al. 2008a; of cardenolides in A. curassavica (Agrawal 2004; Zehnder & Hunter Lefe`vre et al. 2010), we found that, in the absence of aphids, parasites 2007a). As expected (Martel & Malcolm 2004; Zehnder & Hunter produced much lower sporeloads (2.37 times lower) on monarchs 2007b), the rapid population growth of aphids affected the cardeno- reared on the high-cardenolide A. curassavica than on monarchs reared lide concentrations in the high-cardenolide A. curassavica, but not in on the low-cardenolide A. incarnata (Fig. 2a, white bars). Conse- the low-cardenolide A. incarnata, probably because the latter species quently, infected adult monarchs lived much longer (an additional has little room to reduce its cardenolide concentrations below 4.8 days, which is 65% longer) when reared on A. curassavica than on constitutive levels in response to aphid herbivory. A. incarnata (Fig. 2b, white bars). As expected, chemical analyses also confirmed that the A. curassavica plants used in this study had much Aphid effects on the chemistry of the high-cardenolide A. curassavica higher cardenolide concentrations than A. incarnata plants (Fig. 3). Although plants were assigned randomly to treatment groups, A. curassavica plants that were assigned to receive aphids started off Effects of aphid-infested milkweed species on virulence and with lower overall cardenolide concentrations on day 0, and this transmission of monarch butterfly parasites difference was maintained throughout to day 13 (Fig. 3a,b; F = 12.7, P = 0.0008). However, these differences in the overall In contrast to results on aphid-free plants, in the presence of aphids, 1,52 cardenolide concentrations did not affect our results, because the parasites produced similar sporeloads on monarchs reared on the

(a) (b) 6.0 5.8 Without aphids 12 With aphids Figure 2 Effects of milkweed species and the presence of aphids 5.6 on parasite sporeload and virulence. (a) In the absence of aphids, 5.4 parasite sporeload was lower on monarchs reared on the high- cardenolide Asclepias curassavica than on monarchs reared on the 5.2 8 low-cardenolide Asclepias incarnata; when aphids were present, this (sporeload)

10 5.0 milkweed effect disappeared (milkweed species · aphid treatment 4 interaction: F1,96 = 16.91, P = 0.045). (b) Consequently, infected

Log 4.8

lifespan index (day) adult monarch butterflies lived longer when reared on A. curassavica

4.6 Infected monarch adult than A. incarnata in the absence of aphids, but not in the presence 4.4 0 of aphids (milkweed species · aphid treatment interaction: A. curassavica A. incarnata A. curassavica A. incarnata F1,97 = 6.93, P = 0.0098). Bars show mean + 95% confidence Food plant species Food plant species intervals.

2011 Blackwell Publishing Ltd/CNRS Letter Community affects virulence and transmission 457

(a)A. curassavica, day 0 (b) A. curassavica, day 13

) 6.0 6.0 *

–1 Without aphids 1.2 With aphids 1.2

4.0 4.0 0.8 0.8

2.0 2.0 0.4 0.4 * Concentration (mg g

0.0 0.0 0.0 0.0 **

Total Total RT065RT092RT115RT162RT175RT240RT256RT280RT320RT355RT452RT551RT565RT584RT613RT650 RT065RT092RT115RT162RT175RT240RT256RT280RT320RT355RT452RT551RT565RT584RT613RT650 Cardenolide Cardenolide

(c) A. incarnata, day 0 (d) A. incarnata, day 13 0.3 0.3

) 0.4 0.4 –1

0.3 0.2 0.3 0.2

0.2 0.2 0.1 0.1 0.1 0.1 Concentration (mg g

0.0 0.0 0.0 0.0

Total Total RT065RT092RT115RT162RT175RT240RT256RT280RT320RT355RT452RT551RT565RT584RT613RT650 RT065RT092RT115RT162RT175RT240RT256RT280RT320RT355RT452RT551RT565RT584RT613RT650 Cardenolide Cardenolide

Figure 3 Cardenolide concentrations in the high-cardenolide Asclepias curassavica and the low-cardenolide Asclepias incarnata. Cardenolides were measured on day 0 (a and c for A. curassavica and A. incarnata respectively) and day 13 (b and d for A. curassavica and A. incarnata respectively) in plants that received aphids or not on day 0. Asterisks in (b) highlight four cardenolides for which there was a significant interaction between aphid presence and day. The initial differences in concentrations of RT162 and RT256 on day 0 had disappeared by day 13 (day · aphid treatment interaction: F1,51 = 4.45, P = 0.04 and F1,51 = 11.4, P = 0.001 for RT162 and RT256 respectively), whereas RT584 and RT650 increased to higher levels on day 13 in plants without aphids than in plants with aphids (see text for details). Cardenolides are named based on their relative retention time (RT) under reverse-phase HPLC; increasing RTs represent decreasing polarity. Bars show mean + 95% confidence intervals. Note that y-axis scales differ between panels to facilitate interpretation of bar heights.

overall cardenolide concentrations had no effect on parasite sporeload 400 A. curassavica (Fig. 5A; F1,25 = 0.0028, P = 0.96). A. incarnata Aphids significantly changed the overall composition of cardeno- 300 lides in A. curassavica over time. Principle components analysis was used to describe this composition, and together, principle component axes 1–3 (pca1–3) explained more than 60% of the variance in 200 cardenolides (Supporting Information). Of these, the first axis (pca1) showed a significant interaction between day and aphid treatment:

Aphid number pca1 was lower in plants with than without aphids on day 0, but higher 100 in plants with than without aphids on day 13 (day · aphid treatment interaction: F1,51 = 11.27, P = 0.001). Restricting the analysis to day 13 only confirmed that pca1 scores were higher on that day: 0 036912 F1,28 = 5.20, P = 0.030). Interactions between day and aphid treat- Day ment were not significant for pca2 and pca3 (F1,51 = 0.13, P = 0.72 and F = 0.16, P = 0.69). The finding that aphids significantly Figure 4 Aphid population growth. Population growth of Aphis nerii grown on the 1,51 high-cardenolide Asclepias curassavica and the low-cardenolide Asclepias incarnata. increased pca1 between days 0 and 13 demonstrates that they changed Each plant received six aphids on day 0. Data points show mean + 95% confidence the overall composition of cardenolides in the high-cardenolide intervals. A. curassavica.

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(a)Total cardenolide concentration (b) Total cardenolides: pca Figure 5 Relationships between cardenolide concentration and 6.4 parasite sporeload in the high-cardenolide Asclepias curassavica. The overall concentration of cardenolides was not associated with parasite sporeload (a), but the overall composition of cardenolides 5.9 was (b). Furthermore, higher concentrations of the individual cardenolides RT584 (c) and RT650 (d) were associated with lower 5.4 parasite sporeloads. Cardenolide concentrations shown are those

(sporeload) measured on day 13, when monarch caterpillars were infected. 10 4.9 Lines show least-squares regression lines. Note that cardenolide Log data were obtained from plants for a subset of experimental 4.4 only (Table 1), so that numbers of animals in these graphs are lower than those used in Fig. 2. Note that since we found 0369 –4 –3 –2 –1 0 1 no effects of aphids on the cardenolide concentrations in the Concentration (mg g–1) pca1 low-cardenolide A. incarnata, no data are shown for this species. (s) Without aphids; (d) With aphids. (c)RT584 (d) RT650 6.4

5.9

5.4 (sporeload) 10 4.9 Log

4.4 0.0 0.4 0.8 1.2 1.6 0.00.10.20.30.40.5 Concentration (mg g–1) Concentration (mg g–1)

When analysing individual cardenolides, we found that aphids In terms of overall cardenolide composition, we found that higher appeared to mitigate an ontogenetic increase of the concentration of values of pca1 resulted in higher parasite sporeloads (Fig. 5b; cardenolide RT584 (Fig. 3a,b; day · aphid treatment interaction: F1,25 = 8.75, P = 0.007), suggesting that the higher sporeloads in F1,51 = 3.90, P = 0.054), while significantly mitigating an ontogenetic monarchs infected on A. curassavica plants with aphids could be increase of cardenolide RT650 between days 0 and 13 (Fig. 3a,b; explained by their different cardenolide compositions on day 13. day · aphid treatment interaction: F1,51 = 7.87, P = 0.007). As a Similarly, regression analysis showed that higher concentrations of result, A. curassavica plants with aphids had lower concentrations of the individual chemicals RT584 and RT650 were associated with these two chemicals than did plants without aphids on day 13, when lower parasite sporeloads (Fig. 5c,d; F1,25 = 5.41, P = 0.028 and caterpillars were infected with parasites (F1,28 = 7.92, P = 0.009 and F1,25 = 4.85, P = 0.037 for RT584 and RT650 respectively). Because F1,28 = 8.76, P = 0.006 for RT584 and RT650 respectively). the P-values of these regression analyses were relatively high and because the sporeload values were used in both analyses, we carried Aphid effects on the chemistry of the low-cardenolide A. incarnata out a partial Bonferroni correction to ensure that the significance of In the low-cardenolide A. incarnata, total cardenolide concentrations these tests was not the result of carrying out multiple tests. We used and concentrations of RT065 and RT355 decreased between day 0 the web-based statistical program SISA (http://www.quantitatives- and 13 (F1,56 = 29.5, P < 0.0001; F1,55 = 22.5, P < 0.0001; and kills.com/sisa/calculations/bonfer.htm), which accounts for variable F1,56 = 39.6, P < 0.0001 for total, RT065 and RT355 respectively) dependence while correcting for inflated Type I error rates (Garcı´a but aphids did not affect these decreases (day · aphid treatment: 2004). Given a Pearson correlation coefficient of 0.657 (P < 0.0001) F1,54 = 0.005, P = 0.94; F1,54 = 2.38, P = 0.13; and F1,54 = 1.30, between RT584 and RT650 concentrations on day 13, the adjusted a P = 0.26 for total, RT065 and RT355 respectively). Thus, aphids had for each of the two regression analyses is 0.039; hence, we conclude no effect on the cardenolide composition of the low-cardenolide that the negative associations between sporeload and the concentra- A. incarnata. tions of RT584 (P = 0.028) and RT650 (P = 0.037) are indeed significant. In these regression analyses, aphid presence had no further effect on sporeload when cardenolide concentration was Aphid-mediated indirect effects on virulence and transmission of included in the model as a covariate (F = 0.15, P = 0.70 and monarch butterfly parasites 1,24 F1,24 = 0.11, P = 0.74 for analyses with RT584 and RT650 Aphid-mediated indirect effects on the high-cardenolide A. curassavica respectively). These results suggest that the lower concentrations of As shown above, the presence of aphids increased the virulence and RT584 and RT650 cardenolides in A. curassavica plants with aphids transmission potential of parasites in monarchs reared on the high- may explain the higher parasite sporeloads that we observed cardenolide A. curassavica. In addition, aphids changed the overall (Fig. 2a). composition of cardenolides and mitigated an ontogenetic increase of Overall, these results suggest that aphids indirectly increase the two cardenolides in A. curassavica. Here, we tested whether these virulence and transmission potential of monarch butterfly parasites effects on milkweed chemistry can account for the observed changes by decreasing the defensive chemistry of the high-cardenolide in parasite virulence and transmission potential. A. curassavica.

2011 Blackwell Publishing Ltd/CNRS Letter Community affects virulence and transmission 459

Aphid-mediated indirect effects on the low-cardenolide A. incarnata increases in foliar phenolics that then help to protect larvae from As shown above, aphids had no effects on the parasite sporeload of viral infection (Hunter & Schultz 1993). Additionally, studies have monarchs reared on the low-cardenolide A. incarnata, and also did not shown that predatory midges can enhance bacterial parasite affect the defensive chemistry of this species. These results suggest transmission by tearing apart bacteria-filled waterfleas (Ca´ceres et al. that aphids did not indirectly affect parasite virulence and transmission 2009), that predators can suppress the immunity of a herbivorous potential in monarchs reared on A. incarnata. beetle to its parasites (Ramirez & Snyder 2009) and that co-infecting parasite species can affect each otherÕs transmission through modulating host immunity (Graham 2008; Jolles et al. 2008). Our Effects of aphids and milkweed plants on uninfected monarchs study supports these findings, but also goes further by illustrating Overall, uninfected monarchs lived much longer than infected that a fourth species even further removed from the direct host– monarchs (compare Fig. S1, Fig. 2b; F1,128 = 427, P < 0.0001). parasite interaction in the food web may affect a host–parasite There was also a significant three-way interaction between infection interaction by altering the traits – in this case the defensive status, milkweed species and aphid treatment (F1,128 = 10.46, chemistry – of a third species. P = 0.0015). This interaction indicates that the lifespan index of Our results have important implications for understanding host– uninfected monarchs was more strongly decreased by aphids on the parasite dynamics. Theoretical models have indicated that virulence low-cardenolide A. incarnata, whereas the lifespan index of infected and transmission are two key parameters that affect the spread of monarchs was only decreased by aphids on the high-cardenolide parasites in populations and the severity of disease outbreaks A. curassavica (compare Fig. S1, Fig. 2b). (Anderson & May 1991; Hudson et al. 2002). Moreover, a trade-off When restricting the analysis to uninfected monarchs only, we between virulence and transmission is generally expected to select for found that aphids reduced the adult lifespan index of monarchs that parasites with intermediate levels of host exploitation, at which level were reared on both milkweed species (Fig. 2b; F1,32 = 12.8, the costs of exploitation (parasite-induced host mortality) are P = 0.001). Additionally, uninfected monarchs lived longer when balanced by the benefits (between-host transmission) (Anderson & reared on the low-cardenolide A. incarnata than on the high- May 1982; Van Baalen & Sabelis 1995; De Roode et al. 2008b; Alizon cardenolide A. curassavica (Fig. 2b; F1,32 = 12.9, P = 0.001). The et al. 2009). These models generally assume that virulence and interaction between aphid treatment and milkweed species was nearly transmission are under the full control of host and parasite (reviewed significant (F1,33 = 4.15, P = 0.0502), suggesting that aphids more in Restif & Koella 2003) and that other species in the food web do strongly reduced the adult lifespan index of uninfected monarchs not affect these traits. However, our results suggest that other species reared on A. incarnata than on A. curassavica. may have strong impacts on virulence and transmission. In our study, the plants on which monarchs feed as larvae affect these traits, as do the aphids that share this food source with monarchs. How indirect DISCUSSION effects on virulence and transmission affect parasite dynamics would We have shown that the ecological community within which hosts and likely depend on the strength of these effects as well as the parasites interact can have important effects on parasite virulence and environmental heterogeneity in species composition. Theoretical transmission potential. In particular, our results suggest that oleander models will be required to formally predict the ecological and aphids can reduce the levels of some secondary compounds in evolutionary consequences of such indirect effects (e.g. Choo et al. A. curassavica that appear to inhibit a specific parasite of monarch 2003). butterflies. In doing so, aphids can remove the medicinal effects of The parasites of herbivorous are often affected by the plants these milkweeds and increase the virulence and transmission potential upon which insects feed (Cory & Hoover 2006). Such plant effects (sporeload) of monarch parasites. Importantly, our results also show may occur in two ways: either the plant chemicals directly interfere that the indirect effects on virulence and transmission potential caused with the parasite or the plant affects the general health and immunity by a fourth species (oleander aphid) depend on the identity of the of the . The results obtained here suggest that milkweed third species: specifically, aphids have indirect effects when infected chemicals directly interfere with the monarchÕs parasite and that the monarchs develop on the high-cardenolide A. curassavica. In contrast, indirect effect of aphids is mediated through these chemicals. We did aphids have no effect on the virulence and transmission potential of find that aphids reduced the adult lifespan index of uninfected parasites when infected monarchs develop on the low-cardenolide monarchs reared on both milkweed species (Fig. S1), suggesting that A. incarnata, probably because this species has little room to reduce its monarchs that co-occur with aphids are in worse overall health. This cardenolide concentrations below constitutive levels in response to may be due to increased competition for resources, or aphid-induced aphid herbivory. reductions in food quality, such as reduced levels of nitrogen. Our study confirms that species outside the direct interaction However, this reduction in lifespan index did not appear to be related between host and parasite can have indirect effects on host–parasite to disease susceptibility because the lifespan index of infected traits. Most studies to date have shown that third species can have monarchs was only reduced by aphids when they were reared on density-mediated indirect effects on host–parasite interactions, by A. curassavica. Moreover, our results show that the presence of aphids changing the absolute or relative abundance of host species (Mitchell on the high-cardenolide A. curassavica changed its cardenolide et al. 2002; Keesing et al. 2006; Johnson et al. 2008; Borer et al. 2009). composition (Fig. 3) and that these changes were associated with In contrast, studies of trait-mediated indirect effects on parasite-host changes in parasite sporeload and adult lifespan index of infected dynamics are still relatively rare (Raffel et al. 2008, 2010). Where they monarchs (Fig. 5). These results also suggest that any factor that alters have been carried out, such studies have confirmed that indirect the chemical composition of milkweeds may have indirect effects on effects may shape virulence and transmission parameters. For the transmission potential and virulence of the monarch butterflyÕs example, feeding by gypsy moth larvae on oak foliage causes parasite.

2011 Blackwell Publishing Ltd/CNRS 460 J. C. de Roode et al. Letter

As outlined in the introduction, previous studies have found that Choo, K., Williams, P.D. & Day, T. (2003). Host mortality, predation and the the presence of oleander aphids can result in reductions in cardenolide evolution of parasite virulence. Ecol. Lett., 6, 310–315. concentrations of high-cardenolide milkweeds (Martel & Malcolm Cory, J.S. & Hoover, K. (2006). Plant-mediated effects in insect-pathogen inter- actions. Trends Ecol. Evol., 21, 278–286. 2004; Zehnder & Hunter 2007b). Here, we did not find aphid-induced Crawley, M.J. (2002). Statistical Computing: An Introduction to Data Analysis Using S-Plus. reductions in cardenolide concentrations nor did we find a change in John Wiley & Sons, Chichester. overall cardenolide levels. Instead, we found that the presence De Roode, J.C., Pedersen, A.B., Hunter, M.D. & Altizer, S. (2008a). Host plant of aphids prevented a developmental increase in the concentration of species affects virulence in monarch butterfly parasites. J. Anim. Ecol., 77, two particular non-polar cardenolides in the high-cardenolide species 120–126. A. curassavica (Fig. 3). This finding supports the view that overall De Roode, J.C., Yates, A.J. & Altizer, S. (2008b). Virulence-transmission trade-offs cardenolide concentrations of milkweeds may not be fully informative and population divergence in virulence in a naturally occurring butterfly parasite. Proc. Natl Acad. Sci. U.S.A., 105, 7489–7494. of their ecological effects and that it is important to analyse individual De Roode, J.C., Chi, J., Rarick, R.M. & Altizer, S. (2009). Strength in numbers: high compounds (Zehnder & Hunter 2007b). This was also suggested by parasite burdens increase transmission of a protozoan parasite of monarch Fordyce & Malcolm (2000) who found that overall cardenolide butterflies (Danaus plexippus). Oecologia, 161, 67–75. concentrations in milkweed stems with weevil eggs were lower than Fordyce, J.A. & Malcolm, S.B. (2000). Specialist weevil, Rhyssomatus lineaticollis, does those without, but contained more non-polar forms, which are not spatially avoid cardenolide defenses of common milkweed by ovipositing thought to be more toxic. into pith tissue. J. Chem. Ecol., 26, 2857–2874. ´ In conclusion, our results show that species in food webs may Garcıa, L.V. (2004). Escaping the Bonferroni iron claw in ecological studies. Oikos, 105, 657–663. indirectly alter the virulence and transmission parameters of parasites, Graham, A.L. (2008). Ecological rules governing helminth-microparasite coinfec- and that these effects may be mediated through the traits of yet other tion. Proc. Natl Acad. Sci. U.S.A., 105, 566–570. species. Our study of trait-mediated indirect effects complements others Hatcher, M.J., Dick, J.T.A. & Dunn, A.M. (2006). How parasites affect interactions that have illustrated density-mediated indirect effects of community between competitors and predators. Ecol. Lett., 9, 1253–1271. structure on host–parasite interactions. Studying virulence and Helms, S.E. & Hunter, M.D. (2005). Variation in plant quality and the population transmission in a community context will not only increase our dynamics of herbivores: there is nothing average about aphids. Oecologia, 145, 197–204. understanding of the ecology and evolution of parasites, but will also Helms, S.E., Connelly, S.J. & Hunter, M.D. (2004). Effects of variation among plant help in predicting the effects of human-altered communities on the species on the interaction between an herbivore and its parasitoid. Ecol. Entomol., disease risks of humans and wild species alike (Keesing et al. 2006, 2010). 29, 44–51. Hudson, P.J., Rizzoli, A., Grenfell, B.T., Heesterbeek, H. & Dobson, A.P. (2002). The Ecology of Wildlife Diseases. Oxford University Press, Oxford. ACKNOWLEDGEMENTS Hunter, M.D. & Schultz, J.C. (1993). Induced plant defenses breached? Phyto- We thank A. Chiang and N. Lowe for help with the experiment, and chemical induction protects an herbivore from disease. Oecologia, 94, 195– 203. the Emory IHoP journal club as well as four anonymous referees, Johnson, P.T.J., Hartson, R.B., Larson, D.J. & Sutherland, D.R. (2008). Diversity M. Holyoak and K. Lafferty for helpful and constructive comments and disease: community structure drives parasite transmission and host fitness. on previous versions of this manuscript. Funding was provided by Ecol. Lett., 11, 1017–1026. Emory University (to JCdR) and National Science Foundation awards Jolles, A.E., Ezenwa, V.O., Etienne, R.S., Turner, W.C. & Olff, H. (2008). Inter- DEB-0814340 to MDH and DEB-1019746 to JCdR and MDH. actions between macroparasites and microparasites drive infection patterns in free-ranging African buffalo. Ecology, 89, 2239–2250. Keesing, F., Holt, R.D. & Ostfeld, R.S. (2006). Effects of species diversity on REFERENCES disease risk. Ecol. Lett., 9, 485–498. Keesing, F., Belden, L.K., Daszak, P., Dobson, A., Harvell, C.D., Holt, R.D. et al. Abrams, P.A. (1995). Implications of dynamically variable traits for identifying, (2010). Impacts of biodiversity on the emergence and transmission of infectious classifying, and measuring direct and indirect effects in ecological communities. diseases. Nature, 468, 647–652. Am. Nat., 146, 112–134. Koch, R.L., Venette, R.C. & Hutchison, W.D. (2005). Influence of alternate prey on Ackery, P.R. & Vane-Wright, R.I. (1984). Milkweed Butterflies: Their Cladistics and predation of monarch butterfly (Lepidoptera: Nymphalidae) larvae by the mul- Biology. Cornell University Press, Ithaca, NY. ticolored Asian lady beetle (Coleoptera: Coccinellidae). Environ. Entomol., 34, Agrawal, A.A. (2004). Plant defense and density dependence in the population 410–416. growth of herbivores. Am. Nat., 164, 113–120. Lafferty, K.D., Dobson, A.P. & Kuris, A.M. (2006). Parasites dominate food web Agrawal, A.A. & Fishbein, M. (2006). Plant defense syndromes. Ecology, 87, S132– links. Proc. Natl Acad. Sci. U.S.A., 103, 11211–11216. S149. Lefe`vre, T., Oliver, L., Hunter, M.D. & De Roode, J.C. (2010). Evidence for trans- Alizon, S., Hurford, A., Mideo, N. & Van Baalen, M. (2009). Virulence evolution generational medication in nature. Ecol. Lett., 13, 1485–1493. and the trade-off hypothesis: history, current state of affairs and the future. MacNeil, C., Dick, J.T.A., Hatcher, M.J., Terry, R.S., Smith, J.E. & Dunn, A.M. J. Evol. Biol., 22, 245–259. (2003). Parasite-mediated predation between native and invasive amphipods. Anderson, R.M. & May, R.M. (1982). Coevolution of hosts and parasites. Parasi- Proc. R. Soc. Lond., B, Biol. Sci., 270, 1309–1314. tology, 85, 411–426. Malcolm, S.B. (1991). Cardenolide-mediated interactions between plants and her- Anderson, R.M. & May, R.M. (1991). Infectious Diseases of Humans – Dynamics and bivores. In: Herbivores: Their Interactions With Secondary Plant Metabolites (eds Control. Oxford University Press, Oxford. Rosenthal, G.A. & Berenbaum, M.R.). Academic Press, San Diego, pp. 251– Borer, E.T., Mitchell, C.E., Power, A.G. & Seabloom, E.W. (2009). Consumers 296. indirectly increase infection risk in grassland food webs. Proc. Natl Acad. Sci. Malcolm, S.B. & Zalucki, M.P. (1996). Milkweed latex and cardenolide induction U.S.A., 106, 503–506. may resolve the lethal plant defence paradox. Entomol. Exp. Appl., 80, 193– Brower, L.P. & Fink, L.S. (1985). A natural toxic defense system – cardenolides in 196. butterflies versus birds. Ann. N Y Acad. Sci., 443, 171–188. Martel, J.W. & Malcolm, S.B. (2004). Density-dependent reduction and induction Ca´ceres, C.E., Knight, C.J. & Hall, S.R. (2009). Predator-spreaders: predation can of milkweed cardenolides by a sucking insect herbivore. J. Chem. Ecol., 30, 545– enhance parasite success in a planktonic host-parasite system. Ecology, 90, 2850– 561. 2858.

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McLaughlin, R.E. & Myers, J. (1970). Ophryocystis elektroscirrha sp. n., a neogregarine Zehnder, C.B. & Hunter, M.D. (2007b). Interspecific variation within the pathogen of monarch butterfly Danaus plexippus (L.) and the Florida queen Asclepias in response to herbivory by a phloem-feeding insect herbivore. J. Chem. butterfly D. gilippus berenice Cramer. J. Protozool., 17, 300–305. Ecol., 33, 2044–2053. Mitchell, C.E., Tilman, D. & Groth, J.V. (2002). Effects of grassland plant species diversity, abundance, and composition on foliar fungal disease. Ecology, 83, 1713– 1726. SUPPORTING INFORMATION Mouritsen, K.N. & Poulin, R. (2005). Parasites boosts biodiversity and changes Additional Supporting Information may be found in the online community structure by trait-mediated indirect effects. Oikos, 108, 344–350. Packer, C., Holt, R.D., Hudson, P.J., Lafferty, K.D. & Dobson, A.P. (2003). version of this article: Keeping the herds healthy and alert: implications of predator control for Figure S1 Effects of milkweed species and aphids on lifespan index of infectious disease. Ecol. Lett., 6, 797–802. uninfected monarchs. Raffel, T.R., Martin, L.B. & Rohr, J.R. (2008). Parasites as predators: unifying Table S1 Eigenvalues and per cent of variance explained by principal natural enemy ecology. Trends Ecol. Evol., 23, 610–618. component axes 1–16 of the principal components analysis of Raffel, T.R., Hoverman, J.T., Halstead, N.T., Michel, P.J. & Rohr, J.R. (2010). cardenolide concentrations of Asclepias curassavica plants. Parasitism in a community context: trait-mediated interactions with competition Table S2 and predation. Ecology, 91, 1900–1907. Correlations between each factor (cardenolide) and principal Ramirez, R.A. & Snyder, W.E. (2009). Scared sick? Predator-pathogen facilitation component axis of the principal components analysis of cardenolide enhances exploitation of a shared resource. Ecology, 90, 2832–2839. concentrations of Asclepias curassavica plants. Restif, O. & Koella, J.C. (2003). Shared control of epidemiological traits in a coevolutionary model of host-parasite interactions. Am. Nat., 161, 827–836. As a service to our authors and readers, this journal provides Telfer, S., Lambin, X., Birtles, R., Beldomenico, P., Burthe, S., Paterson, S. et al. supporting information supplied by the authors. Such materials are (2010). Species interactions in a parasite community drive infection risk in a peer-reviewed and may be re-organized for online delivery, but are not wildlife population. Science, 330, 243–246. copy edited or typeset. Technical support issues arising from Van Baalen, M. & Sabelis, M.W. (1995). The dynamics of multiple infection and the supporting information (other than missing files) should be addressed evolution of virulence. Am. Nat., 146, 881–910. to the authors. Van Zandt, P.A. & Agrawal, A.A. (2004). Community-wide impacts of herbivore- induced plant responses in milkweed (). Ecology, 85, 2616–2629. Werner, E.E. & Peacor, S.D. (2003). A review of trait-mediated indirect interactions in ecological communities. Ecology, 84, 1083–1100. Editor, Kevin Lafferty Wootton, J.T. (1994). The nature and consequences of indirect effects in ecological Manuscript received 16 September 2010 communities. Annu. Rev. Ecol. Syst., 25, 443–466. First decision made 20 October 2010 Zehnder, C.B. & Hunter, M.D. (2007a). A comparison of maternal effects and Second decision made 7 December 2010 current environment on vital rates of Aphis nerii, the milkweed-oleander aphid. Manuscript accepted 31 January 2011 Ecol. Entomol., 32, 172–180.

2011 Blackwell Publishing Ltd/CNRS Supplementary information Belonging to: De Roode, J.C., Rarick, R.M., Mongue, A.J., Gerardo, N.M. & Hunter, M.D. (2011) Aphids indirectly increase virulence and transmission potential of a monarch butterfly parasite by reducing defensive chemistry of a shared host plant.

without aphids 35 with aphids 30

25

20

15

10

5 Uninfected monarch adult monarch Uninfected lifespan index (days) 0 A. curassavica A. incarnata Food plant species

Figure S1. Effects of milkweed species and aphids on lifespan index of uninfected monarchs. Aphids reduced the lifespan index of monarchs reared on both species, and monarchs lived longer when reared on the low-cardenolide A. incarnata than on the high-cardenolide A. curassavica. Bars show mean + 95% confidence intervals.

Table S1. Eigenvalues and percent of variance explained by principal component axes 1-16 of the principal components analysis of cardenolide concentrations of A. curassavica plants. Principal Percent of Cumulative percent component axis Eigenvalue variance of variance 1 5.00129 31.3 31.3 2 2.90128 18.1 49.4 3 2.06907 12.9 62.3 4 1.38963 8.7 71 5 1.04545 6.5 77.5 6 0.82272 5.1 82.7 7 0.65755 4.1 86.8 8 0.65327 4.1 90.9 9 0.36407 2.3 93.2 10 0.28618 1.8 94.9 11 0.26438 1.7 96.6 12 0.19267 1.2 97.8 13 0.13849 0.9 98.7 14 0.09725 0.6 99.3 15 0.07177 0.4 99.7 16 0.04492 0.3 100

Table S2. Correlations between each factor (cardenolide) and principal component axis of the principal components analysis of cardenolide concentrations of A. curassavica plants. Factor 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 RT065 0.1623 -0.1042 0.3337 -0.1016 0.4325 0.1457 0.252 -0.6933 0.1165 0.2304 0.0427 0.0962 0.0173 0.0201 -0.1008 -0.0075 RT092 0.1188 -0.4001 0.2901 -0.2227 -0.2819 0.0823 -0.1223 0.0198 -0.2004 0.1775 -0.561 -0.1363 0.1503 0.1979 0.104 0.336 RT115 -0.2132 -0.1143 -0.4006 -0.1054 0.4111 -0.2837 0.2718 0.1021 -0.3241 0.2215 -0.1293 0.299 0.1026 0.2554 0.3155 0.0239 RT162 0.2908 -0.3569 0.0531 0.102 0.0328 0.1807 -0.181 0.0294 -0.0285 -0.4027 0.1542 0.6095 -0.1862 -0.0835 0.2905 0.1526 RT175 -0.1579 -0.2961 0.0084 0.1772 0.4936 0.2779 -0.5091 0.2421 0.043 0.2792 0.205 -0.2564 0.1389 -0.0823 0.0153 0.0582 RT240 -0.01 -0.1936 0.4004 -0.4724 -0.0503 -0.2318 0.1867 0.3978 -0.012 0.266 0.4058 0.0679 -0.2568 -0.1233 -0.0679 -0.0606 RT256 0.3557 -0.1654 -0.2628 0.1097 0.069 -0.0112 -0.0652 0.0388 -0.2658 0.2332 -0.329 0.0311 -0.4351 -0.2845 -0.3773 -0.3279 RT280 -0.3666 -0.155 0.1615 0.1748 0.0338 -0.0248 0.1917 0.0286 0.3989 -0.0019 -0.378 -0.0769 -0.2753 -0.3555 0.4406 -0.2033 RT320 0.2194 -0.1634 -0.1776 0.1268 -0.0142 0.5469 0.6373 0.3202 0.1184 -0.0012 0.069 -0.1669 0.0761 0.103 -0.041 0.0695 RT355 -0.1554 -0.2657 0.3278 0.4469 0.11 -0.3066 0.1189 0.1842 0.078 -0.2126 -0.1205 0.1672 0.1678 0.288 -0.4738 -0.1209 RT452 0.1454 -0.4631 -0.2005 -0.0636 -0.2499 -0.1948 0.0125 -0.1819 0.0509 -0.0196 0.1899 -0.1006 0.5241 -0.2059 0.1231 -0.4529 RT551 -0.3049 -0.2858 -0.0585 -0.2565 0.089 0.135 0.0083 -0.1841 -0.2866 -0.5 0.0863 -0.3699 -0.3399 0.2546 -0.0504 -0.1917 RT565 0.3316 -0.1626 -0.1613 0.2346 -0.0578 -0.385 -0.055 -0.1006 0.3256 0.1468 0.1804 -0.2946 -0.359 0.4186 0.1976 0.1668 RT584 -0.2942 -0.2835 -0.3339 -0.0501 -0.0881 -0.1456 0.1039 -0.172 0.1438 -0.0288 0.0668 0.0252 -0.0399 -0.3703 -0.3535 0.5995 RT613 -0.229 -0.0389 0.1682 0.5152 -0.3333 0.079 0.1266 -0.193 -0.5274 0.2806 0.2914 -0.0102 -0.1075 -0.0631 0.1484 0.0482 RT650 -0.3289 -0.102 -0.193 -0.1131 -0.3262 0.3198 -0.1714 -0.0897 0.3146 0.3232 0.0195 0.3793 -0.1241 0.3766 -0.1544 -0.2377