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Animal Behaviour 89 (2014) 155e162

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Animal Behaviour

journal homepage: www.elsevier .com/locate/anbehav

Larval feeding choices in heliconians: induced preferences are notconstrained by performance and host plant phylogeny

Ana Kristina Silva a, Gislene Lopes Gonçalves b,c, Gilson Rudinei Pires Moreira d,*

a PPG Biologia Animal, Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, BrazilbDepartamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazilc Instituto de Alta Investigación, Universidad de Tarapacá, Antofagasta, Arica, ChiledDepartamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

a r t i c l e i n f o

Article history:Received 28 July 2013Initial acceptance 9 September 2013Final acceptance 6 December 2013Available online 3 February 2014MS. number: A13-00618R

Keywords:Heliconiushost plant selectioninsect learningNeotropicsPassiflora

* Correspondence: G. R. P. Moreira, Departamentociências, Universidade Federal do Rio Grande do Su91501-970 Porto Alegre, RS, Brazil.

E-mail address: gilson.moreira@ufrgs.br (G. R. P. M

0003-3472/$38.00 � 2014 The Association for the Stuhttp://dx.doi.org/10.1016/j.anbehav.2013.12.027

Induction of feeding preference, a special type of learning found in the larval stage of herbivorous insects,may be a link between evolutionary changes in host selection behaviour, variation in diet breadth andevolution of host races. However, it has been argued that the phenomenon may be phylogeneticallyconstrained in oligophagous species, which in general use closely related host plants. Also, inductionmight be tied to performance, being more likely to occur in host plants that are more suitable for larvalfeeding. If so, induction would be less likely to promote increase in diet breadth. By conducting reciprocalrearing and using leaf disks and double-choice feeding tests, we explored the influence of these factorson the induction of larval feeding preference in the first and last instars of two oligophagous heliconianbutterflies (Heliconius erato (Linnaeus) and Heliconius ethilla Godart) in relation to the main (five) passionvine species used as host plants by heliconians in southern Brazil. We also determined the effects of hostplants on survivorship, growth rates and size attained in the adult stage for both butterfly species. Forcomparison, we also carried out a phylogenetic analysis of these plants, based on DNA sequencing. Forboth heliconian species, we found that feeding preference could be induced for most host plant speciestested, in the fifth instar in particular, suggesting that habituation is involved in such cases. There was noindication of the existence of host plant phylogenetic constraints. We also found positive responses forinduction on plants that supported poor larval performance. We conclude that induction of larval feedingpreference in these cases was not limited by phylogenetic relatedness among the hosts or by their abilityto support larval performance, which varies in spatial distribution and abundance in the area. We discussthe possible evolutionary consequences of this behavioural phenomenon in these heliconian butterflies.� 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

The existence of induction of feeding preference is usuallyinferred when a host plant that is initially eaten less often is eatenmore often in reciprocally reversed dual-choice feeding tests fortwo rearing plant species. In such cases, a preference is recognizedas having been induced by feeding on that plant (Albert & Parisella,1985; Jermy, Hanson, & Dethier, 1968). Because of difficulties inassigning this behaviour to traditional categories of learning (e.g.habituation and associative learning), it has been considered to be aspecial type of learning: a larval feeding experience effect foundonly among phytophagous insects (Dethier, 1982, 1988; Jermy,1987; Papaj & Prokopy, 1989). The phenomenon is still poorlyknown and its underlying physiological mechanisms are littlestudied. It occurs commonly among lepidopteran larvae, but the

de Zoologia, Instituto de Bio-l, Av. Bento Gonçalves 9500,

oreira).

dy of Animal Behaviour. Published

degree of induction varies among species. The functional implica-tions remain uncertain (Bernays & Weiss, 1996). There are alsostudies showing the reverse effect; that is, feeding on a given hostmakes a caterpillar more likely to prefer a different host (Bernays &Chapman, 1994).

Evolutionary change in host selection behaviour certainly playsa major role in determining both diet breadth and speciation, andbehaviours that lead herbivorous insects to accept novel host plantshave long been recognized as important (e.g. Dethier, 1970; Jermy,1987). Induction of feeding preference is potentially one of theselinks, particularly when herbivores are able to choose amongdifferent host plants to feed on in the wild, and especially if thesechoices are linked to oviposition preferences. In a variable envi-ronment, this plasticity in behaviour has the potential to increaseboth diet breadth and geographical range, when the latter is facil-itated by colonization of a novel host. In turn, it may lead eventuallyto the development of host races (sensu Jaenike & Papaj, 1992),within the genetic assimilation scenario originally described by

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A. K. Silva et al. / Animal Behaviour 89 (2014) 155e162156

Waddington (1953) and in the broader context of phenotypicplasticity (Pfennig et al., 2010; Pigliucci, 2005; West-Eberhard,2003).

In spite of these intriguing possibilities, the induction of feedingpreference has received scant attention in the evolutionary contextof host plant selection. Furthermore, it is thought to be lessimportant when oligophagous insects are involved. This may bepartly because induction of feeding preference has been foundmostly among polyphagous insects, which use a relatively broadtaxonomic spectrum of host plants (for examples in Lepidoptera,see Del Campo & Renwick, 2000; Hanson, 1983; Jermy et al., 1968;Saxena & Schoonhoven, 1982; Ting, Ma, & Hanson, 2002). That is, itis believed that feeding preference is less likely to be inducedamong closely phylogenetically related host plant species. Ac-cording to Boer and Hanson (1984) and Bernays and Chapman(1994), this response could be related to the smaller differencesin phytochemical profiles found between closely related plantscompared to distantly related ones. Furthermore, as suggested bythe results obtained by Dukas and Bernays (2000) and Egas andSabelis (2001), feeding preference would be more likely to beinduced on host plants that provide better nutrition for larvalrearing, and thus would function as a mechanism to reinforce theuse of the most suitable hosts available in a given area. In thepresent study, we experimentally explored these two aspects, bothof which would decrease the importance of induction of feedingpreference as a mechanism of promoting speciation by notfavouring changes in host plant use.

We re-examined the occurrence of induction of feeding pref-erence in the larval stage of heliconian butterflies, which areoligophagous feeders on Passifloraceae (for reviews of the biologyof HeliconiusePassiflora associations, see Benson, Brown, & Gilbert,1976; Brown, 1981; Gilbert, 1991). This phenomenon was firststudied in the PassifloraeHeliconius system by Kerpel and Moreira(2005), who failed to find induction of larval feeding preference.The authors, however, included only one species in their study(Heliconius erato (Linnaeus)) and covered a limited host plantspectrum (two passion vine species). In this study, we added asecond heliconian species (H. ethilla Godart) for comparison, andcovered a much wider range of host plants for both species,including the main host plants used by these heliconian species infar southern Brazil. In addition, we took into account host plantphylogenetic relatedness, by obtaining in parallel a phylogeny forthese passion vines, based on DNA sequences. Also, we evaluatedtheir suitability as hosts for both heliconian species by rearing themon these passion vines under controlled laboratory conditions, andmeasured mortality, growth rates and size attained as adults.

METHODS

Insects and Plants

Larvae used in the experiments came from eggs obtained fromadults of H. erato phyllis (Fabricius) and H. ethilla narcaea Godartthat were field-collected by using butterfly nets, respectively, in theHorto Florestal Barba Negra (HFBN), Barra do Ribeiro Municipality,Rio Grande do Sul State (RS) (30�230S; 51�120W) and the FlorestaNacional de São Francisco de Paula (FLONA), RS (29�240S; 50�220W).Collections were made under IBAMA/ICMBio license number2024629, granted to G. R. P. Moreira. Experiments followed therecommendations of the Animal Care and Use Committee (CEUA) ofthe Federal University of Rio Grande do Sul (UFRGS).

Adults were kept in outdoor insectaries (dimensions2.0 � 2.0 � 2.7 m) maintained at the Zoology Department ofUFRGS, Porto Alegre, RS (30�040S; 51�060W) and fed daily with aseminatural diet (Ferro, 1998). Passiflora misera and Passiflora alata

shoots were placed in plastic bottles, provided with a 50 cm tallwooden frame support and offered for oviposition, respectively, toH. erato and H. ethilla. Eggs obtained were removed from the shootsand placed on petri dishes lined with moist filter paper and kept inthe laboratory under controlled conditions (25 � 1 �C, 14:10 hlight:dark cycle) for incubation.

Seedlings from the host plants tested in this study (Passifloraactinia Hook., P. alata Curtis, P. caerulea L., P. edulis Sims, P. miseraKunth and P. suberosa L.) were collected in different localities in RS,including the municipalities of São Francisco de Paula, Barra doRibeiro, Maquiné (29�350S; 50�160W) and Porto Alegre. They weretransplanted to an outdoor Passiflora garden at the Department ofZoology at UFRGS, received organically enriched soil during trans-planting and thereafter were watered as needed. These species ofpassion vine include the main hosts of these two heliconian speciesin the region, using as references the studies of Périco and Araújo(1991), Périco (1995) and Dell’Erba, Kaminski, & Moreira (2005).

To investigate the extent to which food preferences are phylo-genetically constrained, we used species from the two major cladesof passion vines that occur in southeastern Brazil (subgenera Pas-siflora and Decabola; Mondin, Cervi, & Moreira, 2011) and that wereincluded in the experiments to reconstruct phylogenetic relation-ships. The phylogeny was recovered based on molecular markerspreviously sequenced by Muschner et al. (2003) and Muschner,Zamberlan, Bonatto, and Freitas (2012). DNA sequences fromseven species were used to reconstruct evolutionary relationships.In addition, Mitostemma brevifilis and Paropsia madagascariensis,both members of Passifloraceae but older and distantly related toPassiflora, were incorporated in the analysis to root the tree (fordetails, see Supplementary material, Tables S1, S2). Two majorclades (Passiflora and Decabola) were recovered, with a wideevolutionary divergence from each other (ca. 18%) for a species-level phylogeny (Fig. 1). The mean genetic distances observedwithin the Passiflora and Decaloba clades were markedly different,3% and 9%, respectively. In addition, Passiflora showed two internalclades, one formed by P. alata þ P. edulis and a second includingP. actinia þ P. caerulea. The mean evolutionary distance was 4%between these groups and 3% within groups.

Effect of Food on Survival, Development and Adult Size

Freshly hatched larvae ofH. erato (N ¼ 25) and H. ethilla (N ¼ 30)were placed individually on the terminal bud of shoots. Each Pas-siflora shoot was maintained in a bottle of water, as describedabove, and protected by a thin mesh cloth to contain the larvae(Ferro, 1998). Observations were performed daily, when survivor-ship and moult were recorded, as well as instar length, fromhatching to adult emergence. Shoots were replaced when neededto allow ad libitum feeding.

Forewing length was taken as the distance between the distalend of the radial sector and the origin of the subcostal vein, whichwas measured with callipers (accuracy of 0.02 mm) in newlyemerged adults. During measurements, specimens were gentlyhandled with their wings folded, and were subsequently released.

Larval Feeding Preference

To test for feeding preference, additional larvae of H. erato andH. ethilla were reared on those host plants for which the perfor-mance experiments indicated good survival. To evaluate the exis-tence of preference and respective food plant induction, larvaewere reared on one of the two plant species and then reciprocallytested on each (Fig. 2). Double-choice tests (based on the descrip-tion of Kerpel & Moreira, 2005) were conducted individually forfirst and fifth instars of both H. erato and H. ethilla larvae (varying

55.4

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Passiflora edulis

PassifloraD

ecaloba

Passiflora alata

Passiflora caerulea

Passiflora actinia

Passiflora misera

Passiflora suberosa

Mitostemma brevifilis

Paropsia madagascariensis

CenozoicEocene MioceneOligocene

23.8 5.3

Figure 1. Phylogenetic relationships and divergence times obtained with a Bayesian approach using four genetic markers from six Passiflora species and a related taxon (Mitos-temma). Paropsia was used to root the tree. Numbers above branches represent posterior probabilities. Asterisk indicates posterior probability <0.7.

A. K. Silva et al. / Animal Behaviour 89 (2014) 155e162 157

from 19 to 25 individuals/test). Larvae were tested during themorning of the second day after either hatching (first instar) ormoulting (fifth instar). They were allowed to feed before they wereplaced in the test, with no starvation period.

Larvae were tested for 5 h in plastic pots containing an array ofleaf disks for feeding. We used a cork borer to cut the leaf disks(101.12 mm2 in area), then pierced the disks with micro-entomological pins and distributed them equidistantly, with spe-cies interleaved, in plastic pots containing moistened filter paper(Hanson, 1983). During the tests, the pots were kept undercontrolled abiotic conditions (25 � 1 �C; LD 14:10 h). At thebeginning, larvae were carefully removed from their respectiverearing shoots and placed in the centre of the pot. After the feedingtrial, they were returned to their original plant. Consumption rateswere taken into account for each larval instar during these tests;three and 18 leaf disks per species of Passiflora per larva testedwereoffered for the first and fifth instars, respectively (Rodrigues &Moreira, 1999). Total leaf area per species of passion vine offeredwas adjusted to double the average leaf area consumed by a giveninstar during 5 h to provide a full choice to the larvae throughoutthe experiment.

reared o

preferenc

A

A1 B1

?

Figure 2. Experimental scheme adopted for testing larval feeding choices and changes in fwhen at least twice the damage to one plant (either A or B) was observed in relation to the othan twice the damage relative to the other species were considered as ties. Feeding changesplant A and choosing plant A (¼A1) minus the number of larvae reared on plant B but choosand choosing plant B (¼B2) minus the number of larvae reared on plant A but choosing pl

The remaining leaf disk portions were glued to a paper sheet todetermine food consumption. The area consumedwasmeasured byplacing the leaf disks on graph paper at the end of each feeding trialand counting the number of square millimetres corresponding tothe missing leaf area. Feeding trials were considered as choiceswhen at least twice the damage to one host plant was observed inrelation to another one in a given test. We considered feeding trialsas ties when this pattern was not observed (Thomas, 1987). Thisprocedure was adopted to reduce bias in the area measurement,because the consumption per leaf disk was generally low.

Finally, we considered positive responses to induction in pref-erence to occur when feeding changes in the reciprocal choice testswere significantly greater in favour of the plants used for rearing(Jermy et al., 1968).

Statistical Analysis

The results for larval survivorship for both species were orga-nized into contingency tables and compared using Fisher’s exacttest, followed by Bonferroni correction. The results for developmenttime and adult size were evaluated for homogeneity of variance of

n

e for

B

A2 B2

?

ood selection as a function of rearing plant. Feeding trials were considered as choicesther plant in a given test. Cases where leaf disks of neither Passiflora species had morewere measured as follows: in favour of A ¼ A1 � A2 (i.e. the number of larvae reared oning plant A (¼A2)); in favour of B ¼ B2 � B1 (i.e. the number of larvae reared on plant Bant B (¼B1)).

A. K. Silva et al. / Animal Behaviour 89 (2014) 155e162158

data, assessedbyBartlett test, and for normal distributionof thedataby the KolmogoroveSmirnov test. For H. erato, the data for devel-opment time were normally distributed, and therefore were testedby one-way ANOVA, followed by Tukey multiple tests. The dataobtained for H. ethilla were not normally distributed, and werecompared by nonparametric KruskaleWallis test, followed byDunn’smultiple comparison tests. Data on the size of adults for bothspecies were evaluated by one-way ANOVA, followed by Tukeymultiple comparison tests. Feeding preference in double-choicetests was compared by bilateral sign tests, adopting the null hy-pothesis that the number of choicesmade in favour of the plant usedfor rearing was equal to that in relation to the other plant tested inthe pair, disregarding the number of ties (Table 1). Overall data on

Table 1Feeding selection by first- and fifth-instar larvae of two heliconian species in relationto Passiflora plants, on double-choice leaf disk tests, when they were reared on oneof two plant species and then reciprocally tested on each

Heliconian Rearingpairs

Test pairs

First instar Fifth instar

H. erato edu ties mis edu ties misedu 3 6 11* 5 4 11*mis 2 3 15* 0 2 18*

edu ties cae edu ties caeedu 16* 2 2 13* 5 2cae 16* 3 1 6 6 8

edu ties sub edu ties subedu 8 5 7 16* 2 2sub 7 6 7 5 9 6

sub ties cae sub ties caesub 11* 6 3 11* 6 3cae 8 8 4 5 4 11*

mis ties cae mis ties caemis 15* 5 0 19* 1 0cae 13* 5 1 11* 4 5

mis ties sub mis ties submisy 30* 2 8 38* 1 1suby 18 7 15 30* 9 1

H. ethilla edu ties mis edu ties misedu 7 2 11 8 6 6mis 6 3 11 2 6 12*

edu ties ala edu ties alaedu 13 2 9 9 8 8ala 5 3 17* 4 8 13*

edu ties act edu ties actedu 7 2 11* 15* 1 4act 8 3 9 7 2 11

edu ties cae edu ties caeedu 3 4 13* 7 6 7cae 8 2 10 1 4 15*

mis ties ala mis ties alamis 14 2 9 6 8 11ala 5 3 17* 2 6 17*

mis ties act mis ties actmis 15* 5 0 11* 9 0act 19* 0 1 12 13 5

mis ties cae mis ties caemis 7 5 8 8 10 2cae 9 6 5 3 7 10*

ala ties act ala ties actala 6 3 11 14* 5 1act 15* 2 3 14* 1 5

ala ties cae ala ties caeala 9 7 4 11* 5 4cae 12* 4 4 2 6 12*

act ties cae act ties caeact 11 4 9 10* 10 3cae 2 6 17* 4 9 12*

Data show the number of larvae falling into each preference category. The ‘tie’category corresponds to cases where leaf disks of neither Passiflora species hadmorethan twice the damage relative to the other species. act: P. actinia; ala: P. alata; cae:P. caerulea; edu: P. edulis; mis: P. misera; sub: P. suberosa.

* Significant difference for sign tests, a ¼ 0.05.y Data from Kerpel and Moreira (2005).

feeding changes as a functionof rearingplantwere comparedwithininstars through unilateral Wilcoxon signed-ranks tests for onesample, adopting median �0 as the null hypothesis. These para-metric and nonparametric tests followed the criteria described byZar (1999) and Conover (1980), respectively. Unless noted other-wise, measurements are given as means þ SE, and alpha ¼ 0.05.

RESULTS

Performance

Of seven passion vine species used to feed the larvae of H. erato,five allowed survivorship greater than 60% (P. misera, P. edulis,P. suberosa, P. actinia and P. caerulea), and survivorship did not differstatistically between these species (Fig. 3a). For larvae on P. alata,mortality was 100%, and occurred in the first instar. In this case, thelarvae ingested a negligible amount of food and lived less than 1day.

Survivorship of H. ethilla larvae did not differ when reared onP. misera, P. edulis, P. alata, P. actinia or P. caerulea, reaching morethan 90% on these plants (Fig. 3b). Survivorship was significantlylower when they were fed with P. suberosa.

Among those passion vine species where survivorship wasgreater for H. erato, development time was shortest on P. edulis,which significantly differed from the development time on P. miseraand P. suberosa. Development time was intermediate on P. actiniaand longest on P. caerulea (Fig. 4a). For H. ethilla, development timewas shortest on P. misera, intermediate on P. edulis and P. actinia,and longest on P. alata and P. caerulea (Fig. 4b).

Adult size of H. erato was significantly greater on P. edulis andP. suberosa than on P. caerulea. It was intermediate on P. misera and

100 a

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Figure 3. Survivorship of (a) Heliconius erato (N ¼ 20) and (b) H. ethilla (N ¼ 30) fromlarval hatching to adult emergence, when reared on different passion vines (Passifloraspp.) under laboratory conditions (25 � 1 �C; LD 14:10 h). Within heliconian species,bars with the same letter were not significantly different (for both species, Fisher’smultiple comparison exact tests, with Bonferroni correction, a ¼ 0.0083).

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ta

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tinia

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erulea

27

26

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24

23

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Figure 4. Development time of (a) Heliconius erato (N ¼ 20) and (b) H. ethilla (N ¼ 30)from larval hatching to adult emergence, when reared on different passion vines(Passiflora spp.) under laboratory conditions (25 � 1 �C; LD 14:10 h). Within heliconinespecies, bars with the same letter were not significantly different (H. erato: one-factorANOVA: F4,88 ¼ 31.24, P < 0.01, followed by Tukey multiple comparison tests, a ¼ 0.05;H. ethilla: KruskaleWallis test: H4 ¼ 15.74, P ¼ 0.0034, followed by Dunn multiplecomparison tests, a ¼ 0.05).

42

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tinia

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ulis

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ta

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tinia

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erulea

Figure 5. Adult size of (a) Heliconius erato (N ¼ 20) and (b) H. ethilla (N ¼ 30) whenreared from larval hatching to emergence on different passion vines (Passiflora spp.)under laboratory conditions (25 � 1 �C; LD 14:10 h). Within each heliconine species,bars with the same letter were not significantly different (one-factor ANOVA: H. erato:F4,92 ¼ 6.23, P < 0.01; H. ethilla: F4,132 ¼ 19.46, P < 0.01, followed by Tukey multiplecomparison tests, a ¼ 0.05).

A. K. Silva et al. / Animal Behaviour 89 (2014) 155e162 159

P. actinia (Fig. 5a). For H. ethilla, adult size was larger on P. edulisthan on P. caerulea, and intermediate on the other plants (Fig. 5b).

Preference

In the double-choice tests withH. erato, first-instar larvae rearedon P. edulis and then tested for preference with respect to P. edulisand P. caerulea chose the former species; first-instar larvae alsopreferred P. edulis when reared on P. caerulea, but not in the fifthinstar, which showed no preference. When tested on P. edulis andP. suberosa, P. edulis was chosen by fifth instars when reared onP. edulis. When reared either on P. suberosa or P. caerulea, larvaepreferred the plant species used for rearing, except in the first instarfor P. caerulea, where there was no choice. Passiflora misera wasalways the preferred plant, even when larvae were reared on otherpassion vines (P. edulis, P. caerulea and P. suberosa), with theexception of larvae fed with P. suberosa in the first instar.

In the double-choice tests with H. ethilla, fifth-instar larvaewhen reared on P. misera, P. alata or P. caerulea preferred theseplants over P. edulis. Rearing on P. edulis did not influence choicesregarding P. misera, P. alata, P. actinia and P. caerulea, except for thelatter in the first instar. In all remaining cases therewas influence ofthe rearing plant at least in the fifth instar, with the exception of thecomparison between P. alata and P. actinia, where the former wasthe preferred plant in both cases.

In these tests, overall feeding changes were reciprocally signif-icant in favour of the rearing plant for the fifth instar (Fig. 6b, d), butnot for the first instar (Fig. 6a, c), in both heliconian species.

DISCUSSION

Induction of Feeding Preference

Our results clearly show that feeding preference can be inducedin the larval stage of H. erato and H. ethilla in relation to five speciesof passion vines used as host plants in southern Brazil. Therefore,preference in feeding choices by these two butterfly species are notall innate, but can be learned by experience while feeding on theseplants. We attribute the failure of Kerpel andMoreira (2005) to findan induction of preference in H. erato larvae to the small number ofhost plants tested (only two). Their results may also reflect theinclusion of P. misera, a host plant species that was always chosenby H. erato larvae when reared on any of the other species testedhere, which presumably indicates that these larvae have an innatepreference for this particular plant. As pointed out by Bernays andWeiss (1996), for unknown reasons, there is variation in the degreeof response among species that show positive results for inductionof larval feeding preference. The data presented herein corroboratethis statement, suggesting that the number of host plants testedmay alter the conclusions about the existence of induction offeeding preference and should be taken into account in this kind ofstudy in the future.

Strong positive responses for induction of feeding preferencewere obtained only during the fifth larval instars for the two heli-conian species. As suggested by Bernays and Weiss (1996), induc-tion of larval feeding preference may be related to the mechanismof ‘imprinting’ in the first instar and linked to ‘habituation’ in thelatter stages. Thus, although both behavioural mechanisms mightbe involved, we infer that in these butterflies in particular, habit-uation might be the main mechanism driving induction of larvalfeeding preference, and this possibility should be further explored.

100

A1–A2: W+= 15, W–= 0, P = 0.03*

A1–A2

B2–B1

act - P. actinia

ala - P. alata

cae - P. caerulea

edu - P. edulis

mis - P. miserasub - P. suberosa

B2–B1: W+= 13, W–= 2, P = 0.09

A1–A2: W+= 21, W–= 0, P = 0.02*B2–B1: W+= 15, W–= 0, P = 0.03*

A1–A2: W+= 26.5, W–= 28.5, P = 0.54B2–B1: W+= 20.5, W–= 15.5, P = 0.37

A1–A2: W+= 44, W–= 1, P < 0.0001B2–B1: W+= 55, W–= 0, P < 0.0001

(a)

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edu-cae edu-sub sub-cae mis-cae mis-sub

edu-mis edu-ala edu-act edu-cae mis-ala mis-actRearing and test pairs

mis-cae ala-act ala-cae act-cae

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Figure 6. Feeding changes by first and fifth larval instars of (a, b) Heliconius erato and (c, d) H. ethilla, as a function of rearing plant (Passiflora spp.), in double-choice feeding testsunder laboratory conditions (25 � 1 �C; LD 14:10 h). A1 � A2 and B2 � B1: feeding changes in favour of plants A and B, respectively (for details, see Fig. 2); Wþ and W�: Wilcoxonsigned-ranks test, positive and negative scores, respectively. Asterisks indicate significant differences, a ¼ 0.05.

A. K. Silva et al. / Animal Behaviour 89 (2014) 155e162160

Phylogenetic Constraints

Our findings do not support the hypothesis for the existence ofan inverse correlation between likelihood of induction in feedingpreference and host plant phylogenetic relatedness (Wasserman,1982). This is very important in the context of evolution of dietbreadth and speciation of herbivorous insects, since host plantshifts have occurred with greater frequency between closelyrelated plants than between distantly related ones (Janz & Nylin,1998). However, while studying the induction of feeding

preference in the larval stage of Manduca sexta (Sphingidae) inseveral solanaceous species, Boer and Hanson (1984) found positiveresponses only for comparisons between plants belonging todifferent genera. Conversely, negative results for induction whilecomparing closely related plants were found by Chew (1980) withlarvae of Pieris napi (Pieridae); in this case, several cruciferousspecies with similar glucosinolate profiles were used (Boer &Hanson, 1984). In the present study, in contrast, we demonstratedits occurrence within a single host plant genus (Passiflora) foroligophagous heliconian species. Furthermore, there was no

A. K. Silva et al. / Animal Behaviour 89 (2014) 155e162 161

indication that host plant relatedness influenced the results, evenon the finest phylogenetic scale (i.e. within groups). For H. ethilla inparticular, we found positive responses in relation to host plantspecies belonging not only to different subgenera (with geneticdistance ca. 18%) but also between closely related lineages, withinclades at the subgeneric level (for example, P. caerulea versusP. actinia; mean genetic distance of 4%). Thus, the existence of anegative correlation between the likelihood of induction in feedingpreference and host plant phylogenetic relatedness should be re-examined. It may not be applicable to the HeliconiusePassiflorasystem in particular, since even among the closely related passionvine species tested here there exist differences in chemical profiles,for example regarding flavonoids and saponins (Birk, Provensi, &Gosmann, 2005), which are widely recognized as playing animportant role in host plant use by herbivorous insects, includingmembers of Lepidoptera (Gershenzon & Croteau, 1991; Hartmann,1991). An extensive survey of lepidopterans is needed to deter-mine whether this negative correlation between likelihood of in-duction in feeding preference and host plant phylogeneticrelatedness suggested in the literature holds true for most species(i.e. is a pattern). The negative correlations found so far may haveresulted from the small number of systems examined. Bernays andWeiss (1996) argued that M. sexta is the only species that has beentested for the existence of induction of larval feeding preferenceusing a large number of host plant species and different larvalinstars.

Correlation with Performance

Although all the plants tested in the present study are used ashosts and support satisfactory development (e.g. Dell’Erba et al.,2005; Périco, 1995; Périco & Araújo, 1991), P. misera andP. suberosa are the most abundant species, the most preferred andused as hosts and are also those that are most suitable as hosts forH. erato in central Rio Grande do Sul (Kerpel & Moreira, 2005;Rodrigues & Moreira, 2002). In this part of the state, H. ethillaoviposition is highest on P. alata, followed by P. edulis and P. actinia;whereas it does not occur on P. suberosa (Périco, 1995). Passifloraalata and P. edulis are not native to RS, andwere introduced recentlyto the region for cultivation purposes (Koehler-Santos, Lorenz-Lemke, Salzano, & Freitas, 2006; Mondin et al., 2011). As shownherein, most H. ethilla larvae do not develop on P. suberosa, inagreement with the findings of Miranda (1997). Studies conductedby Périco (1995) and Miranda (1997), however, did not include thenortheast region of RS, where P. alata and P. edulis are uncommon(Moreira, Ferrari, Mondin, & Cervi, 2011), and where H. ethilla pri-marily uses P. actinia, followed by P. caerulea (Moreira, 2011).Although in this study H. ethilla performed satisfactorily onP. misera, we have indications that this heliconian butterfly does notuse this plant in RS or elsewhere (Beccaloni, Viloria, Hall, &Robinson, 2008). Host preference may be maladaptive, as is espe-cially likely with respect to recently introduced plants (e.g. Karowe,1990; Keeler & Chew, 2008). This should be further studied, since asimilar finding was obtained by Bianchi and Moreira (2005) forDione juno Cramer, another heliconian species; that is, feeding onP. misera leads to satisfactory performance in the laboratory butagain, there is no indication that this plant is effectively used underfield conditions. Other factors, such as the presence of natural en-emies and competitors, may determine the use of a given plant byherbivorous insects, and in the case of heliconian butterflies, alsothe presence of conspecific eggs and larvae (Benson et al., 1976;Mugrabi-Oliveira & Moreira, 1996; Rodrigues, Kaminski, Freitas, &Oliveira, 2010; Singer, Rodrigues, Stireman, & Carriére, 2004).

Thus, our results on performance are in concordance with pre-vious findings related to the success of H. erato and H. ethilla on host

plants used in the study area. We interpreted these findings in thecontext of diet plasticity, a particular characteristic inherent to theseheliconian species; H. erato and H. ethilla use at least 30 and 25passion vine species, respectively, in the Neotropical region, andshow some of the widest diet breadths in the group (Beccaloni et al.,2008). By having this characteristic and thus using a variety of pas-sion vine species, which may differ in their suitability as larval foodandvary in abundance among localities, these species havebeen ableto extend their geographical ranges, which are among the largest inthe genus (see Rosser, Phillimore, Huertas,Willmott, &Mallet, 2012).

On the other hand, our results also do not support the argumentthat larval feeding preference is more likely to be induced amongplants that are more suitable as larval food. As demonstrated byPérico and Araújo (1991) andMiranda (1997), and also confirmed inthe present study, P. caerulea shoots are of low nutritional quality aslarval food for both H. erato and H. ethilla, leading to a longerdevelopment time and smaller size in the adult stage in both cases.However, the larvae were induced to feed on this passion vinespecies in preference to species that ranked higher with respect tolarval development and size, for example in relation to P. suberosafor H. erato and to P. misera for H. ethilla. This may be the underlyingmechanism by which shifts from the most suitable host plantspecies to suboptimal, but more abundant, host plant species occurin the field, as in the case of P. caerulea. This passion vine species iswidespread in the region, occurring in sympatry with the hostplants that are most preferred and used by both H. erato andH. ethilla in central RS, and that were tested in the present study.More importantly, P. caerulea is also common in northeastern RS,where natural grassland vegetation forms a mosaic with AraucariaForest formations, and where H. erato and H. ethilla both occur.Furthermore, it is the only passion vine species that is commonlyfound in the fragmented, southern gallery forests located in theopen grasslands of the Pampa biome, and thus is the passion vinespecies with the southernmost geographical distribution in theNeotropics (Argentinian province of Buenos Aires), where only twoheliconian butterflies,H. erato and Agraulis vanillae (Linnaeus), havebeen recorded (Moreira et al., 2011).

General Conclusion and Possible Evolutionary Consequences

In contrast to the findings of a previous study, we demonstratedhere that larval feeding preference can be induced in heliconianbutterflies. Also, induction in this case was not limited either byphylogenetic relatedness among host plants or by larval perfor-mance while feeding on these plants.

In summary, we infer that the ecological plasticity in host plantuse by these two heliconian butterflies is mediated, at least in part,by learning in the larval stage, which would induce the use of lesssuitable host plants from a nutritional perspective when they aremore abundant than the optimal plants in a given locality (sensuJaenike & Papaj, 1992). This would result first in broadening thediets and spatial distributions of these species, since the distribu-tion of passion vines is not geographically uniform, and would inturn lead to local specializations in host plant use. We are now inthe process of studying this effect, if any, on the initiation of raceformation in these heliconian butterflies.

Acknowledgments

We are grateful to the Instituto ChicoMendes de Conservação daBiodiversidade for allowing us to carry out part of the study atFLONA/São Francisco de Paula. We thank Sidia Maria Callegari-Jacques (UFRGS) for her assistance on statistical analyses. We areespecially grateful to two anonymous referees for their valuablecomments and suggestions, which have substantially improved the

A. K. Silva et al. / Animal Behaviour 89 (2014) 155e162162

manuscript. Thanks are also due Janet W. Reid and Kris Bruner forediting the text. The financial support for this study came in partfrom a CNPq Master’s Fellowship granted to A. K. Silva. G. L. Gon-çalves and G. R. P. Moreira were supported by CNPq grants (156153/2011-4 and 309676/2011-8, respectively).

Supplementary Material

Supplementary material for this article is available, in the onlineversion, at http://dx.doi.org/10.1016/j.anbehav.2013.12.027.

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