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Animal Behaviour 79 (2010) 689–698

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Animal Behaviour

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

Avoiding plants unsuitable for the symbiotic fungus: learning and long-termmemory in leaf-cutting ants

N. Saverschek a, H. Herz b,1, M. Wagner a, F. Roces a,*

a Behavioural Physiology and Sociobiology, Biocenter, University of Wurzburg, Germanyb Smithsonian Tropical Research Institute, USA

a r t i c l e i n f o

Article history:Received 9 July 2009Initial acceptance 27 August 2009Final acceptance 29 November 2009Available online 12 January 2010MS. number: 09-00468

Keywords:Atta colombicaaversive learningdelayed rejectionexperienceforaginghost plant selectionleaf-cutting antmemoryPanama

* Correspondence: F. Roces, Behavioural PhysiologyUniversity of Wurzburg, Am Hubland, D-97074 Wurz

E-mail address: roces@biozentrum.uni-wuerzburg1 H. Herz is at the Smithsonian Tropical Research In

Ancon, Republic of Panama.

0003-3472/$38.00 � 2009 The Association for the Studoi:10.1016/j.anbehav.2009.12.021

Leaf-cutting ants are polyphagous herbivores that, despite their catholicity, show distinct preferences inthe substrate choice for their symbiotic fungus. We investigated to what extent avoidance learning andlong-term memory for plant unsuitability underlie foraging responses in the field. First, the acceptabilityof 10 rarely harvested plant species was tested on field colonies located in two different habitats, wherethe tested plant species were either present or not. Colonies in the habitat where the plant speciesoccurred avoided all species on first encounter, suggesting previous experience with them. Colonieswithout the plant species in their habitat, however, first accepted, but then avoided four of them whentested after 24 and 48 h. Such an avoidance response towards previously acceptable leaves could beexperimentally induced by infiltrating acceptable leaves with a fungicide (cycloheximide) not detectableto the ants, but harmful to the symbiotic fungus, indicating that workers learn to reject plants that havedetrimental effects on the fungus, but not on themselves. To determine how robust memory for plantunsuitability was, workers from field colonies were offered the previously avoided, yet untreated plantevery 2 weeks, and its acceptance monitored in the long term. It took up to 18 weeks until foragersharvested the plant again, indicating the involvement of robust long-term avoidance learning in foragers.We argue that the harvesting pattern observed in field colonies largely depends on the workers’ foragingexperience, essential in a highly diverse environment where both leaf availability and quality varythroughout the year.� 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Leaf-cutting ants, like other herbivores, are influenced byvarious factors in their choice of host plants, and physical andespecially chemical plant characteristics play a major role (Stradling1978). In addition, several leaf-cutting ant species also seasonallyavoid many of their regular hosts (Fowler & Robinson 1979),probably because of inherent changes in the chemical compositionof leaves over time. Whatever the underlying causes, leaf-cuttingant colonies regularly have to search for potential host plants.

Foraging decisions on the suitability of substrates are verycomplex as leaf-cutting ants do not harvest for themselves, but fortheir symbiotic fungus. Adult workers obtain more than 90% oftheir energy requirements from the plant sap of the harvestedplants (Bass & Cherrett 1995), but the fungus garden represents thesole food source of the developing brood. The ants’ plant

and Sociobiology, Biocenter,burg, Germany..de (F. Roces).stitute, P.O. Box 0843, Balboa,

dy of Animal Behaviour. Publishe

preferences are therefore expected to be partially driven by therequirements of the fungus.

Besides the direct acceptance or rejection of plants at the har-vesting site upon the detection of palatable or unpalatablecompounds (Cherrett & Seaforth 1970; Hubbell et al. 1983), hostplant selection may also be influenced by the workers’ previousexperience with the plants. For instance, foragers’ decisions may beinfluenced by the odour of plant fragments carried by nestmatesalong the trail or inside the nest (Roces 1990, 1994; Howard et al.1996). Conversely, repeated offerings of certain plant species maylead to their delayed rejection (Knapp et al. 1990). Such delayedrejection implies avoidance learning by the foragers, which mayoccur because some leaf compounds are harmful to the ants thatingest them (e.g. noxious plant sap), thus leading to rejection, forinstance, via the involvement of postingestive taste aversion, orbecause the harvested substrate is deleterious to the fungus yetharmless for the ants, so that workers react to changes in fungusperformance by discontinuing the harvesting of such material(Knapp et al. 1990). Additionally, foraged material may be furtherassessed based on the physical resistance after its carriage into the

d by Elsevier Ltd. All rights reserved.

N. Saverschek et al. / Animal Behaviour 79 (2010) 689–698690

colony, thus being either processed into the fungus garden or dis-carded (Camargo et al. 2003).

With a fungicide undetectable by the ants, but harmful to thefungus, Ridley et al. (1996); North et al. (1999) and recently Herzet al. (2008) demonstrated the involvement of the symbiotic fungusin the process of delayed avoidance of host plants. Workers initiallyincorporating baits or leaves containing the fungicide into thefungus garden stopped harvesting these substrates in response totheir deleterious effects on the fungus, even though the substrateswere harmless to the ants. Hence, a new potential host plant caneither be rejected without delay by the ants at the cutting sitebecause of the presence of unpalatable compounds (e.g. Hubbellet al. 1983) or rejected after a delay because of either strongphysical resistance or the harmful effects on the fungus after itsincorporation (Ridley et al. 1996; North et al. 1999; Camargo et al.2003; Herz et al. 2008).

We used two different approaches to determine to what extentavoidance learning and memory for plant unsuitability for thefungus might influence diet selection under natural foragingconditions in the field. First, we examined whether experience-mediated avoidance responses to plants is a phenomenon thatactually occurs with native plant species in colonies in the field. Forthat, we tested the acceptance or rejection of 11 plant species in antcolonies from two different habitats, where the plant species to betested were either present or not. Comparison between the twohabitats allowed us to explore the potential role of previousexperience on the subsequent acceptance of natural plants. Such anapproach was further extended by presenting colonies from thetwo habitats with the 11 plant species simultaneously in a choiceexperiment, so as to determine the ants’ rank order of plant pref-erences and potential experience-based changes.

As a second approach, avoidance of previously accepted plantswas experimentally induced in mature field colonies, by presentingthem with acceptable leaves infiltrated with a fungicide notdetectable to the ants, as in previous studies (Ridley et al. 1996;Herz et al. 2008). When delayed avoidance was shown by foragerson the trail where the harmful substrate was harvested, weinvestigated whether workers from other trails also becameexperienced and showed avoidance, and how long memory for theharmful plant species lasted under field conditions.

Note that our experimental approaches using large field colo-nies did not address acquisition or retention performance at theindividual level using controlled learning paradigms, as in recentstudies on ant learning (Dupuy et al. 2006; Kleineidam et al. 2007;Josens et al. 2009). Since workers were not individually marked, weexamined learning abilities and memory as a colony-wideresponse. This kind of ‘colony memory’ probably goes beyond thesum of individual memories, because experienced individualsmight influence the response of naıve workers, as previously sug-gested (Jaffe et al. 1985). The situation is even more complexbecause avoidance learning classically applies to stimuli associatedwith a contingent negative effect on the individual. Leaf-cuttingants are known to learn the negative effects of a given plant on thesymbiotic fungus, and not on themselves, as indicated above(reviewed in Herz et al. 2008). Whatever the underlying mecha-nisms leading to the association, in the present study we refer toavoidance learning and long-term memory as colony-wideresponses.

METHODS

Study Sites

The study was conducted in Panama between November 2002and August 2003. Mature colonies of the leaf-cutting ant Atta

colombica (Guerin) were located on Barro Colorado Island (BCI;9�090N, 79�510W) and in Gamboa (9�070N, 79�420W) in the CanalArea. The 10 colonies investigated on BCI were in a species-richtropical moist forest of late secondary status. In Gamboa, the 10study colonies were in an open park-like area, with predominantlygrass and herbs and scattered fruit and ornamental trees. The sitewas chosen as the surrounding vegetation was scarce and speciespoor. We verified that all tested plant species were not present inthe foraging area of the colonies used in Gamboa.

Influence of Learning on Plant Acceptance

We tested whether delayed avoidance of native plant speciesoccurs in field colonies, and if plant acceptance is influenced byprevious experience of workers. Therefore we compared theacceptance of 11 native plant species by colonies in the two habitatsmentioned above. The tested plant species were chosen froma long-term study on the foraging ecology of an A. colombica colonyon BCI which included lists of all plant species that had been har-vested over the period of 1 year and of all plant species thatoccurred in the foraging area (Wirth, 1996; Wirth et al. 2003). Fromthese lists, we chose plant species that were rarely harvested, thatis, species with a high probability of being ‘delayed rejected’ ifprevious harvesting experience, and therefore avoidance learning,is important in determining diet selection. Seven plant species thathad been harvested once, twice or never during that 1-yearobservation were assayed (see Results). We also tested Hymenaeacourbaril, a species known to be avoided by Atta cephalotes becauseof deterrent compounds (Hubbell et al. 1983). Finally, we used twospecies that occur in the habitat, but are not cut (personal obser-vations). For comparison, we also tested one frequently harvestedattractive plant species, Spondias mombin.

On BCI, all tested plant species are rather common (Croat 1978)and we assumed that workers from the 10 experimental colonieshad previous experience with them. In Gamboa, none of the testedplant species was growing in the foraging area of the 10 experi-mental colonies; therefore those colonies were considered naıve.Henceforth BCI is referred to as p-habitat (tested plant speciespresent in the habitat) and Gamboa as a-habitat (tested plantspecies absent in the habitat).

The procedure for the field tests for plant acceptability was asfollows. On the first day, we conducted a pick-up test for accept-ability, then the colonies were provided with a given amount of thetested plant species, so as to allow workers to gain experience withit, and we tested acceptance again on the 2 following days usingsimilar pick-up tests.

To control for the physical properties of leaves, such as leaftoughness, that may influence acceptance, leaves were offered asdiscs (diameter 7 mm) that were collected by ants without the needfor cutting. All leaf material offered was collected from three indi-vidual plants per species and at least three leaves per individual,partly to take into account the chemical and physical interindi-vidual variability. Each plant species was tested on three colonies ineach location. The 11 plant species were tested individually.

We knew from preliminary observations that workers ofA. colombica show one of three behavioural responses when facedwith leaf discs on or on the side of the foraging trail: a forager (1)takes the leaf disc to the nest (acceptance), (2) ignores the leaf disc(passive avoidance) or (3) clears it off the trail (active avoidance).

We tested each plant by presenting 100 leaf discs on an activetrail and recording intake and trail clearing as a measure ofacceptability until either the leaf discs were gone or 30 min hadpassed. If at least one leaf disc was accepted on the first day, thecolonies received leaf discs of the tested species equivalent to about1% of their daily intake (between 350 and 600 leaf discs depending

N. Saverschek et al. / Animal Behaviour 79 (2010) 689–698 691

on colony size). This amount was chosen as laboratory experimentshave shown that a small percentage of their daily intake is sufficientto cause a reaction (personal observation). None of the coloniesharvested the tested plant species naturally on any of their trails atthe time of the experiments.

Each pick-up test was conducted 30 m away from the nestentrance on the side of one randomly chosen active foraging trail.Before a given plant species was tested, we conducted a pick-up testwith 100 sugared oats. As sugared oats (sprayed with a 10% sucrosesolution) are a highly palatable substrate of consistent quality, theywere used as a measure of general foraging activity. As foragingactivity varies between colonies and over time, intake and trail-clearing rates of each tested plant were expressed as a percentagerelative to the number of sugared oats accepted before eachexperiment.

To estimate the daily harvest of the colonies, we counted therefuse deposition rate for 5 min after each experiment. The numberof refuse particles removed from the nest within 24 h correlatesstrongly with the total daily number of harvested plant fragmentscarried into the nest. Whereas the number of ants carrying frag-ments into the nest varies with the time of day, the diurnal refusedumping rate is relatively constant. Daily harvest activity couldtherefore be calculated according to Herz et al. (2007).

An additional experimental series, using simultaneous presen-tation of all 11 plant species, explored the effect of experience onthe rank order of acceptance. A preference test was initially con-ducted on the first day to evaluate acceptability and to establisha rank order of preferences. At the end of this test, colonies receivedabout 100 leaf discs of each of the tested species. We consideredthis sufficient to cause a possible change in acceptability of theplant on the next day. Twenty-four hours later, we tested workersfrom the investigated colonies again to see whether experiencewith the foraged discs led to a change in the rank order of accep-tance of the plant species.

One leaf disc of each of the 11 tested species and one sugared oatwere presented simultaneously along an active foraging trail(Hubbell & Wiemer 1983). They were placed about 5 cm apart alongboth sides of the trail. As soon as the ants removed a disc, it wasrecorded and replaced with a disc of the same species to maintainconstant and equal availability of all disc types. The positioning ofthe leaf discs was randomized every 15 min to exclude possiblepositioning effects (Howard 1987, 1990). Intake and trail-clearingevents were recorded. The experiment ran for 45 min and wasrepeated the following day. The leaf discs were offered on the samesection of the same trail on both days. The tests were conductedabout 10 m away from the nest entrance. At an average walkingspeed of 1.0–1.3 m/min (Lewis et al. 1974; Cherrett, 1989), it isunlikely that an ant could have completed a round trip more thanonce during a single 15 min period (Howard 1987). Again, thesugared oat was used to standardize the intake and trail-clearingrates of the different colonies, as indicated above. We carried outthree replicates in each location on colonies different from the onesabove. None of the colonies harvested the tested plant speciesnaturally on any of their trails at the time of the experiments.

Delayed Avoidance

We experimentally induced delayed avoidance of a highlyacceptable plant substrate by presenting leaves previously infil-trated with a fungicide on a single foraging trail, and exploredwhether workers from other trails also became experienced overtime, that is, whether the avoidance responses were restricted tothe place of first exposure to the treated leaves, or whether allforagers avoided the previously treated plant during foragingindependent of its location.

The plant was offered on all trails, but only one received plantmaterial treated with a fungicide. We conducted the pick-up testsfor 3 consecutive days to check for changes in the level of accept-ability on each trail over time. We used Stigmaphyllon lindenianum,a common, well-accepted host plant species (Wirth et al. 2003;personal observations).

We treated leaf discs with an aqueous solution of the fungicidecycloheximide (CHX; Sigma–Aldrich, Deisenhofen, Germany),which is undetectable by the ants but has been proven to be a potentfungicide to the attine fungus in the laboratory (Ridley et al. 1996;North et al. 1999; Herz et al. 2008). To maintain leaf-specific prop-erties such as odour, surface characteristics and toughness, the leafinternal airspace was infiltrated with a CHX solution (3 mg CHX/mlH2O; see Herz et al. 2008). The leaf discs were briefly rinsed and thensprayed with a 30% sucrose solution to disguise possible changes insubstrate quality over time (Fowler & Robinson 1979).

We investigated 10 colonies, which differed in size and numberof active foraging trails. On the day before the experiment, eachtrail was offered a small amount (see below) of untreated leaf discsto test for general acceptability. On the day of the experiment (day0), each trail of every colony received the same amount of untreatedleaf discs as the day before, except for one randomly chosen trail(henceforth ‘experimental trail’), on which fungicide-treated leafdiscs were offered. As harvesting activity can vary considerablyfrom one day to the next (Wirth et al. 2003), the number of treatedleaf discs offered in this experiment was based on the size of thecolony, as estimated experimentally (see below). To havea comparable level of fungicide-treated leaves incorporated intothe field colonies of different sizes, we offered 10 leaf discs per 400foragers, which equalled about 0.2–0.8% of the harvest on the day ofthe experiment. Leaf discs were offered 5 m away from the nestentrance on the side of the trail, and we confirmed that all theoffered leaf discs were taken into the nest.

We estimated colony size from the number of foragers, by usinga capture–recapture method. At least 500 laden foragers per trailwere marked with a dot of enamel varnish (Revell, Elk GroveVillage, IL, U.S.A.) on their thorax. A preliminary 3-day observationperiod of marked ants in the laboratory showed no deleteriouseffect of the paint: no noticeable change in behaviour could beobserved, and no mortality occurred. Marked ants were kept ina box for at least 15 min before release, to ensure that the paint wasdry and the solvents were evaporated. Twenty-four hours later, thesame number of foragers as marked the day before were collectedand examined for the presence of marking. We used the modifiedLincoln index (Porter & Jorgensen 1980) to calculate the number offoragers of each colony, which allows an estimation of the totalpopulation size within the nest. In Atta sexdens laboratory coloniesforagers make up less than 10% of the total number of workers(Wilson 1980), and in field colonies of A. cephalotes foragersaccount for about 6% (Lewis et al. 1974).

From day 1 on, that is, after the incorporation of the treated leafdiscs, pick-up tests for acceptance were performed with untreated,sugared leaf discs on each trail, for 3 consecutive days. We offered100 sugared leaf discs 5 m away from the nest entrance on the sideof all colony trails for 30 min, and counted pick-ups. Workersaccepting leaf discs were collected off the trail before entering thenest with their load to avoid the incorporation of the previouslyrejected material into the colony.

Long-term Memory

To investigate how long memory for the unsuitable plant specieslasts once avoidance responses were observed, tests for acceptancewere performed at 2-week intervals in five of the 10 colonies used inthe previous experiment, until the colonies accepted the formerly

N. Saverschek et al. / Animal Behaviour 79 (2010) 689–698692

treated plant species S. lindenianum again. Intake rate of untreatedleaf discs was recorded from the very beginning, and trail-clearingrate was only recorded from the 6th week on. Only the treatmenttrail of the colony was tested according to the experimental proce-dure described above. Note that any observed variation in accep-tance over time could a priori be the result of seasonal changes inplant palatability, and not necessarily an effect mediated by thefungus. To control for this possibility, experiments with the five fieldcolonies started at different times over 4 months (November–February; Fig. 1). As all five experimental colonies readily acceptedS. lindenianum before the treatment, this acceptance served asa control to show the unchanged and high palatability of thesubstrate over time. In addition, acceptance tests were also con-ducted on an independent control colony that had never been pre-sented with fungicide-treated S. lindenianum leaves, starting ata later date and carried out also every 2 weeks for 8 weeks (Fig. 1).

RESULTS

Influence of Learning on Plant Acceptance

Intake and trail-clearing rates were used as a sensitive measurefor palatability. Figure 2 summarizes all observed responses acrossthe tests, in all colonies over all days, to provide a general picture ofthe response sensitivity. In a given test, not all workers necessarilyshowed the same response to the offered leaf discs, either intake orremoval (trail clearing). As a consequence, the outcome of each testranged from high intake rates and no trail clearing, thus indicatinghigh acceptance, to no intake and high trail-clearing rates, denotingstrong avoidance. No or low intake rates were accompanied byeither strong or no trail-clearing responses, and vice versa.

All tested plant species could be accepted, immediately avoidedor avoided after successive exposures. To illustrate these patternsover time, Fig. 3 shows examples of three plant species that wereaccepted, immediately rejected or delayed rejected for coloniesfrom both the p-habitat and the a-habitat. Spondias mombin(Fig. 3a) was always accepted on all days in both habitats, and notrail clearing occurred. Desmopsis panamensis, on the other hand,was immediately rejected by all colonies, irrespective of the habitat(Fig. 3b). Foragers from all colonies showed active avoidance, whichmeans almost no intake and a very marked trail-clearing behaviouron all tested days. Hymenaea courbaril was actively rejected byforagers from colonies in the p-habitat (Fig. 3c). They showedalmost no intake and trail-clearing behaviour on all 3 days, witha slightly increasing rejection over time. Foragers from colonies inthe a-habitat, conversely, accepted H. courbaril well on day 1, withnearly no trail clearing, but rejected it on the two following tests ondays 2 and 3. The rejection increased slightly from hardly any intake

A

B

C

D

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1

Experimentalcolonies

Control colony

November December January Feb

Figure 1. Schedule of experimental and control colonies on Barro Colorado Island (BCI) for2003. : Experimental colonies treated with the fungicide CHX; : Experimental colonie

and little trail clearing on the second day to marked rejection withstrong trail clearing on the third day.

Table 1 summarizes the results for all 11 plants tested foracceptance in colonies from both habitats, with presence orabsence of the plant species to be tested. To obtain comparativemeasures, the two behavioural responses recorded were combinedand are presented as a standardized palatability index (PIs), definedas standardized intake rate minus standardized trail-clearing rate.The highly palatable plant species S. mombin, as expected andshown above in Fig. 3a, was well accepted by colonies from bothhabitats. The PIs for this species ranged between 0.20 and 0.38depending on the day of the experiment and the habitat (Table 1).Comparisons between the responses of colonies from the twohabitats when presented with the 10 other, less-attractive plantspecies clearly indicated the occurrence of experience-baseddelayed avoidance. Avoidance, both immediate and delayed, wasmanifested as a very low to nonexistent intake and a marked trailclearing, resulting mostly in negative PIs. Whereas colonies fromthe p-habitat rejected the 10 tested plant species during the firsttest on day 1, as well as on the following days, colonies from thea-habitat initially accepted four of the 10 species (Hymenaea, Inga,Tetragastris and Trichilia), but rejected them on the following 2 daysin a way similar to the colonies in the p-habitat (Table 1, habitat-related rejection species). On day 1, their PIs values were signifi-cantly higher than those of colonies from the p-habitat (statistics inTable 1).

The effect of experience on plant acceptability was also evident inthe additional experiment with simultaneous presentation of the 11plant species. Ranking of the tested plants occurred and changed overtime as a result of experience in colonies from the a-habitat (Fig. 4).Leaf discs of Hymenaea, Inga, Tetragastris and Trichilia were acceptedon day 1, but significantly avoided the following day, thus leading toa general shift in the observed ranking (Fig. 4a). Only two species,Spondias and Miconia, showed a positive PIs on day 2, while the othernine plants were all clearly avoided. Colonies in the p-habitat, on theother hand, showed almost the same preference pattern on both daysof the experiment, that is, the accepted and the rejected plant specieswere assessed similarly on day 2. Only a significant increase inrejection was observed between day 1 and day 2 for Tetragastris andInga (Fig. 4b). The rank order of the tested plant species was almost thesame in both locations on the second day. As in the a-habitat, Miconiawas also accepted on both days of the experiment, in contrast to theresults of the individual tests (Table 1).

Delayed Avoidance

The 10 experimental colonies had two to five active foragingtrails and were of different sizes. Based on the results of the

May Juneruary March April

the experiment on long-term retention of induced rejection. November 2002 to Junes before treatment; ,: Control colony.

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Figure 3. Examples of field acceptance tests of three plant species with colonieslocated in habitats with (p-habitat) and without (a-habitat) the tested plant species.Tests were conducted for 3 days each. Intake (black, positive bars) and trail clearing(white, negative bars) were recorded over 30 min, or until all offered leaf discs hadbeen picked up. Intake and trail-clearing rates of each tested plant were standardizedand expressed as a percentage relative to the number of sugared oats accepted beforeeach experiment. Data are mean � SE with N ¼ 3 colonies for each plant species tested.(a) Spondias mombin: acceptance in both habitats; (b) Desmopsis panamensis: imme-diate rejection in both habitats; (c) Hymenaea courbaril: delayed rejection in a-habitat.

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Figure 2. Trail-clearing rate as a function of intake rate, both expressed as the numberof leaf discs collected over 30 min. Each point represents one of 198 acceptance tests.Eleven plant species were tested on six different colonies over 3 consecutive days. Theinsert shows the data at low intake rates more clearly.

N. Saverschek et al. / Animal Behaviour 79 (2010) 689–698 693

capture–recapture method, two small colonies with 2500–5300foragers, four medium-sized colonies with 13 400–41 200 foragers,and four large colonies with 75 500–137 700 foragers wereinvestigated.

Figure 5 summarizes the results of the acceptance tests with allcolonies over 3 days. Avoidance responses occurred on all trailswith a marked dynamic over time. The intake rates were stan-dardized as percentages of the intake rates on the treatment day,when there was no rejection of the offered plant. One day after theintake of fragments treated with fungicide, acceptance of thefragments was significantly different between the treatment trailsand the adjacent trails. Colonies showed a strong decrease in theintake rate of the treatment trail (about 10% of previous days’intake), but the adjacent trails on average still showed moderateacceptance (20–40%). On days 2 and 3 a nearly complete rejectionof the substrate was observed on all trails.

Long-term Memory

Figure 6 shows the acceptance of the tested plant after the singleexposure to the fungicide-treated leaves, throughout 18 weekswith tests using untreated leaves every 2 weeks. After the initialintake of leaf discs of S. lindenianum treated with fungicide and theobserved rejection 2 days later, very low intake of the untreatedsubstrate could be observed for approximately 12 weeks (Fig. 6a). Inthe 16th and 18th weeks after the treatment, colonies started toaccept the substrate again. To take their size and general foragingactivity into account, intake rates were standardized as percentagesof the intake rates on the treatment day. All colonies clearly avoidedthe substrate for 12 weeks accepting only 0–5% of their originalintake. From the 14th to the 18th week, intake increased fromaround 10% to 50–60% of the colonies’ intake on the treatment day(Fig. 6b).

Resumption of acceptance in the last weeks of the experimentwas characterized by the occurrence of two events, intake and trailclearing, as previously indicated in Fig. 3. While some workers wereobserved to pick up the discs and carry them to the nest, othersremoved the fragments and cleared the trail. Both behaviouralevents were quantified, from week 14 until week 18, and thepalatability index was calculated to combine them. Figure 7 shows

that after 14 weeks colonies still strongly avoided the untreateddiscs, with high trail-clearing rates that resulted in a negativepalatability index. There was a significant difference between thepalatability index of weeks 14 and 18. After 18 weeks, even thoughthere were still a few trail-clearing events in three of the fivecolonies, the palatability indices of the treated colonies were highand reached the same level as that of the control colony.

DISCUSSION

Influence of Learning on Plant Acceptance

Evidence of the involvement of previous experience on plantacceptability has already been described as ‘delayed rejection’ inseveral leaf-cutting ant species under various conditions. In thelaboratory, colonies of Acromyrmex octospinosus have shown

Table 1List of the 11 plant species tested over 3 consecutive days for acceptance or avoidance (N¼three colonies per plant species and habitat)

Species p-Habitat a-Habitat

Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

AcceptanceSpondias mombin 0.26�0.07 0.38�0.16 0.38�0.09 0.30�0.19 0.20�0.10 0.39�0.16

Immediate rejectionDesmopsis panamensis �0.11�0.07 �0.06�0.02 �0.05�0.01 �0.03�0.01 �0.03�0.01 �0.02�0.01Hiraea grandifolia �0.13�0.07 �0.07�0.03 �0.04�0.01 �0.06�0.02 �0.02�0.00 �0.01�0.01Miconia argentea 0.01�0.00 0.00�0.00 0.00�0.00 0.00�0.00 0.00�0.00 0.00�0.00Randia armata �0.50�0.18 �0.16�0.03 �0.15�0.02 �0.08�0.05 �0.05�0.02 �0.03�0.01Sorocea affinis �0.27�0.14 �0.13�0.05 �0.07�0.02 �0.00�0.00 �0.05�0.01 �0.03�0.01Trema micrantha �0.02�0.01 �0.02�0.02 �0.04�0.02 �0.03�0.02 �0.04�0.04 �0.02�0.03

Habitat-related rejectionHymenaea courbaril �0.01±0.03 �0.03�0.04 �0.07�0.05 0.10±0.04 �0.01�0.01 �0.04�0.03

Between habitats on day 1: P<0.05Inga goldmanii 0.03±0.02 �0.02�0.01 �0.04�0.02 0.30±0.05 �0.11±0.03 �0.06�0.01

Between habitats on day 1: P<0.001Day 1–day 2 in a-habitat: P<0.0001

Tetragastris panamensis �0.32±0.11 �0.15�0.03 �0.13�0.02 0.14±0.10 �0.04�0.03 �0.05�0.03Between habitats on day 1: P<0.001

Trichilia tuberculata �0.02±0.07 �0.06�0.01 �0.04�0.03 0.20±0.07 0.06±0.03 �0.01�0.00Between habitats on day 1: P<0.05Day 1–day 2 in a-habitat: P<0.05

Plants are grouped according to the observed behaviour of the ants. Pick-up rates are shown as standardized palatability index (PIs ¼ standardized intake rate�standardizedtrail-clearing rate). Negative values indicate that the rate of trail clearing exceeded the rate of intake. The response of ants to the last four plant species depended on thehabitat. They were immediately rejected in the p-habitat, and rejected after a delay in the a-habitat. Statistically significant differences between habitats or days are indicatedin bold. Statistics were performed using a general linear model followed by a Fisher’s least significant difference test.

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delayed rejection of some plant species, but the mechanisms involvedremained elusive (Knapp et al.1990). Experimentally induced delayedrejection has been shown towards orange granules containingcycloheximide (CHX) in the laboratory with A. sexdens rubropilosa(North et al. 1999) and in the field with A. cephalotes (Ridley et al.1996), and leaves treated with CHX were also delayed rejected bylaboratory colonies of Acromyrmex lundi (Herz et al. 2008).

By comparing the responses of field A. colombica colonies fromtwo different habitats where the tested plant species were presentor absent, we showed that delayed avoidance of several naturalplant species occurs under field conditions. Colonies from thehabitat where the tested plant species were absent showed delayedrejection of four of 10 plant species. Considering that we testeda representative sample of species that were known to be ‘poorly’accepted, it seems that leaf-cutting ants immediately reject about60% of supposedly unsuitable species, whereas experience medi-ates the rejection of the other 40%. To what extent the fungusgarden is involved in the observed delayed aversive response stillremains open. Several authors have pointed out that substratesharvested by leaf-cutting ants are not necessarily the most suitablefor the fungiculture (Hubbell & Wiemer 1983; Hubbell et al. 1983;Powell & Stradling 1986, 1991). As foragers harvest plants tosupport their symbiotic fungus, suitability is difficult to define(Knapp et al. 1990). Foragers might reject suitable substrates fortheir fungus because they contain unpalatable compounds for theants. Conversely, they might accept unsuitable substrates at firstencounter because the unsuitable compounds are unknown orundetectable, and workers have no previous experience with them.

One of our tested plant species, H. courbaril, has been the subjectof several previous studies on host plant selection of leaf-cuttingants. It contains terpenoids that are potent fungicides(caryophyllene epoxide and caryophyllene), and are poisonous insmall doses to the ants’ fungus (Hubbell et al. 1983; Howard et al.1988). Nevertheless leaf-cutting ants harvest H. courbaril regardlessof their fungicidal compounds (Hubbell et al. 1983; this study). Fourreasons were suggested why leaf-cutting ants might harvestH. courbaril: (1) fungicide is sequestered in leaf pocket resin sacksand therefore may be less concentrated on the surface, (2) as leaves

were offered as discs, workers had insufficient contact with leaf sapto identify chemical cues, (3) a lack of cutting costs might make lesspalatable substrates more acceptable and (4) the fungicidalcompounds may vary between plant species (Hubbell et al. 1983).The present study shows that lack of experience might be animportant reason for the occasionally observed harvest of H. cour-baril. Workers from colonies without experience with the plantreadily accepted leaf discs on the first day of the experiment andrejected the substrate on the following days, whereas workers fromexperienced colonies immediately rejected the leaf discs, possiblyreflecting the colonies’ history with the offered species. Rahbe et al.(1988) showed an acquired rejection of yams by A. octospinosus andother authors have demonstrated the effects of prior experience onthe subsequent pattern of resource use in many phytophagusinsects (Papaj & Prokopy 1989; Szentesi & Jermy 1990). Knapp et al.(1990) also pointed out that their laboratory colonies of A. octo-spinosus showed delayed rejection only to plants unlikely to beencountered in their natural environment.

Several factors influence the acceptability of substrates by colo-nies and account for variation between colonies of the same species.Availability of highly preferred host plant species (Hubbell & Wiemer1983), trail activity and palatability of the simultaneously harvestedspecies on the experimental trail are only some of them. Our findingssupport Fowler’s (1982) results with Acromyrmex landolti fracticornisthat suggest that fungal substrate preferences are environmentallyinduced, probably as a result of early experience of workers.

The observed change in the ranking positions of several plantspecies on the second day in the a-habitat suggests that feedingexperience alters the relative ranking of resources (Saxena &Schoonhoven 1982; Deboer & Hanson 1984). Not only avoidancelearning, but also experience gained through appetitive learningcan change the relative acceptance of species. For instance, workersfrom field colonies of A. colombica preferred the one of two palat-able species that they had encountered and accepted the day before(Howard et al. 1996). Considering the role of previous experience indiet selection, it is not surprising that other studies found differ-ences between colonies in preference rankings of simultaneouslypresented, acceptable resources (Howard 1987). Therefore our

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Figure 4. Field preference tests of 11 plant species over 2 days with colonies located in habitats (a) without (a-habitat) and (b) with (p-habitat) the tested plant species. Intake andtrail clearing were recorded for 45 min. Intake and trail-clearing rates were standardized and a palatability index was calculated (¼ standardized intake rate minus standardizedtrail-clearing rate). Data are mean � SE, with N ¼ 3 colonies per habitat. To comply with requirements of normality, all values were arcsine transformed prior to statistical analyses.Statistics were performed using a general linear model (time as predictor variable, no interactions tested) followed by a Fisher’s least significant difference test. *P < 0.05; **P ¼ 0.01;***P < 0.001; a-habitat: Hymenaea: F1,4 ¼ 24.54, P ¼ 0.016; Trichilia: F1,4 ¼ 85.56, P ¼ 0.0008; Tetragastris: F1,4 ¼ 23.61, P ¼ 0.010; Inga: F1,4 ¼ 15.84, P ¼ 0.013; p-habitat: Tetragastris:F1,4 ¼ 19.16, P ¼ 0.012; Inga: F1,4 ¼ 13.38, P ¼ 0.021. Residuals were normally distributed.

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results emphasize the significance of multiple palatability testsover time, to make a valid statement on the host plant range ofA. colombica specifically and probably leaf-cutting ants in general.

Miconia argentea was surprisingly well accepted in the rankingexperiment, in contrast to the previous experiment when it wasoffered alone. A reason for this could be the reference material withwhich M. argentea was offered. Animals generally seem to rankresources in their foraging area and appear to concentrate theirforaging effort on the best items (MacArthur & Pianka 1966;Schoener 1969; Wirth et al. 2003). Within the given choice among11 species of low palatability, M. argentea seemed to be the secondbest option next to S. mombin. Sampling behaviour and intake ofa broad variety of different host plant species by leaf-cutting antscan reflect the lack of a highly palatable resource and therefore theneed for catholicity in their choice of substrates (Krebs 1979).However, it can also be a sign of a colony’s lack of experience.

Delayed Avoidance

As indicated above, experience-dependent avoidance of naturalplant species was observed, although the involvement of the

fungus remained elusive. Clear-cut evidence arose from theexperiments using leaves of S. lindenianum treated with thefungicide cycloheximide. On most of the trails with fungicide-treated leaf discs, a strongly decreased intake within 24 h wasobserved, whereas on the adjacent trails intake was still moderateafter 1 day. These results were similar to those by Ridley et al.(1996) with A. cephalotes in the field, suggesting a lack of infor-mation spread within the nest and/or high trail fidelity of theforagers. Within 2–3 days though, almost complete rejection of thepreviously treated plants was observed.

The observed time delay between the rejection observed at thetreatment trail and that on adjacent trails could hypothetically beaccounted for by considering the internal distribution of theincorporated fragments, without necessarily invoking activeinformation transfer among workers. Several studies with variousAtta species have shown that after incorporation, plant fragmentsare usually distributed evenly throughout the colony within a timewindow ranging from 24 to 48 h (Forti & Silveira Neto 1989; Pretto& Forti 2000; Moreira et al. 2003). Once the plant fragments aredistributed among the available nest chambers, it may in additiontake several hours to incorporate them into the fungus garden, and

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Figure 5. Relative intake rate of untreated Stigmaphyllon lindenianum leaf discs over 3days, based on the intake of the treatment day (day 0). Data are mean � SE with N ¼ 10colonies. All trails of each field colony were tested. Since colonies varied in theirnumbers of adjacent trails, the mean intake rate of all adjacent trails per colony wascalculated. Intake ¼ no. of leaf discs accepted in 30 min. Standardized intake -¼ intakeday after treatment/intaketreatment day. To comply with requirements of normality,all values were arcsine transformed prior to statistical analyses. ANOVA for repeatedmeasurements: time: F2,27 ¼ 13.0, P < 0.001; treatment: F1,27 ¼ 10.6, P < 0.01; time*-treatment: F2,27 ¼ 3.7, P < 0.05. Residuals were normally distributed. Different letters(a, b) indicate significant differences (P < 0.05).

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Figure 6. Long-term acceptance of Stigmaphyllon lindenianum leaf discs after an initialexperimentally induced rejection using leaves treated with fungicide. N ¼ 5 coloniesfor each habitat. (a) Intake rate (no. of leaf discs/30 min) over 18 weeks; (b) stan-dardized intake rate ¼ intake rateday after treatment/intake ratetreatment day. Data aremedian, quartiles and range without outliers.

N. Saverschek et al. / Animal Behaviour 79 (2010) 689–698696

until changes in the fungus become identifiable for workers (Herzet al. 2008). It can be argued that as soon as the substrate iseventually evenly distributed throughout the colony, all availableforagers, experienced or naıve (Herz et al. 2008), might be able toassess the unsuitability of the substrate because of its effects on thefungus, and avoid collecting such a substrate accordingly. In addi-tion, weak trail fidelity could contribute to the occurrence ofcolony-wide aversive responses because workers change trails overtime. In fact, Porter & Bowers (1982) showed with A. colombica thatapproximately 30% of media workers changed trails within 24 h.These percentages seemed to depend strongly on colony size,varying between 15 and 45%, and were observed to stay consistentover 3 days (Wagner 2004).

Long-term Memory

Tests over a period of 20 weeks showed that all field coloniesresumed acceptance of S. lindenianum after 18 weeks. Laboratorycolonies of A. octospinosus rejected yellow yams for 38 days afterthe last contact (Rahbe et al. 1988), and laboratory colonies ofA. cephalotes rejected orange granules with cycloheximide for up to30 weeks (Ridley et al. 1996). In A. lundi, rejection of cycloheximide-treated leaves was maintained for at least 9 weeks when incorpo-ration of the previously unsuitable plant species was precluded(Herz et al. 2008).

Retention in the field was surprisingly long, considering thata colony harvests several different substrates each day (Wirth et al.2003), that is, workers still recognize and reject one specificresource over a prolonged period of time. Our experimentalprocedure prevented the occasional worker that accepted the leafdisc to carry it back into the nest. Therefore the avoidance of theunsuitable leaves could not be reversed by the incorporation ofsuitable leaves of the same species. This means that the reoccur-rence of acceptance after 3–4 months, as observed, depended on

the time passed since the initial incorporation of the substrate, andnot upon new experience gained in the meantime.

Even though the tested colonies were of different sizes, and theoffered amounts of fungicide-treated substrate accounted fordifferent but very low percentages of their daily harvest (0.2–0.8%),all colonies started to reaccept the substrate after a similar timespan. It is yet unknown why workers resume harvesting of theformerly unsuitable plant after such a long period. Hypothetically,once the changes in the fungus garden caused by the unsuitableplant are no longer detectable, foragers should start accepting theplant again if repeated assessment of the conditions in the fungusoccurs. It has been shown that fungicide-induced changes in thefungus, or the identity of the plant fragment on which the fungusgrows, are no longer detectable by naıve ants after 2 days (Herzet al. 2008). In addition, the turnover of the whole fungus gardenhas been estimated to take about 7 weeks (Weber 1972; Powell &Stradling 1986, 1991), even though turnover rates might vary basedon leaf availability (Bass 1997). Yet, the avoidance response in thepresent field experiments lasted more than twice as long. Workersfrom laboratory colonies accepted initially treated plant materialafter 9 weeks if no reversal learning was possible, but showedacceptance after 3 weeks when ‘forced’ to feed on untreated leavesof the previously treated plant species (Herz et al. 2008).

As mentioned above, foraging workers still avoid the plantspreviously unsuitable for the fungus at a time when the fungusgarden contains no trace of either the substrate or the harmful

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Figure 7. Long-term acceptance of Stigmaphyllon lindenianum leaf discs in the lastperiod, before complete reacceptance occurred. Palatability index (PI) for weeks 14–18:(intake minus trail-clearing events)/30 min. Data are median, quartiles and rangewithout outliers. N ¼ 5 colonies for weeks 14 and 16 and N ¼ 4 colonies for week 18.Friedman test: c2 ¼ 8, P ¼ 0.018; Wilcoxon test for multiple comparisons of pairedsamples (weeks 14 and 18): D ¼ 8; P < 0.05. The range of PI of the control colony isrepresented by the grey horizontal bar (PI ¼ 53–80.5). Different letters (a, b) indicatesignificant differences (P < 0.05).

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effect anymore. A more likely explanation for the duration ofrejection and the eventual acceptance of the treated substratemight be the turnover of the population of foragers, as suggested byRidley et al. (1996). Unfortunately, hardly any research has beendone on the life span of workers in general and that of foragers inparticular. Workers of several Atta species have been estimated tolive for 1–2 years (Weber 1972) and some evidence indicates thatolder workers may become foragers (Fowler et al. 1986). However,Porter & Bowers (1982) only estimated a life span of 2–3 weeks forforagers of A. colombica based on a short-term field experiment,where they marked ants and monitored changes in their frequencyon the trails. Foragers of A. capiguara seem to live for 2–3 months(Fowler et al. 1986). That leaves us speculating that, since theduration of the avoidance response roughly matches the life span offoragers of Atta foragers, lifetime individual memory may underliethe observed responses.

Assuming that a number of experienced foragers would die overthe test period of 18 weeks, and naıve foragers would join theforaging column at a given rate every day, a hypothetical linearincrease of acceptance over time should occur. Such a patternwould result as far as each worker decides on its own whethera fragment is suitable or not. However, our results (Fig. 7) indicatethat workers did not gradually start foraging on S. lindenianumagain. Instead, resumption of acceptance occurred from oneacceptance test to the next, in which foragers took leaf discs back tothe nest again. Such a rather sudden change in acceptance might bean indication of workers influencing each other during the collec-tive harvesting activity. It is tempting to speculate that experiencedforagers may directly influence naıve workers by precluding thecollection of unsuitable plant fragments. Accordingly, colony-widememory may be represented by the sum of individual memoriesplus the interactions among nestmates. Over time, however, withincreasing numbers of naıve ants joining the foraging column anddecreasing numbers of experienced foragers because of death,naıve workers would make up the majority of foragers on the trail.

Even if a few foragers still remember the previous experience witha certain plant, the naıve foragers might overrule them, thusleading to a large incorporation of the previously unsuitablesubstrate. Once the previously unsuitable substrate has beenbrought into the nest again, reversal learning can occur (Herz et al.2008).

Even though we did not address acquisition or retentionperformance at the individual level using controlled learningparadigms, as in recent studies on ant learning (Dupuy et al. 2006;Kleineidam et al. 2007), our experiments clearly highlight theinvolvement of avoidance learning during food choice in leaf-cutting ants. In our opinion, the present results go beyond classicalstudies on conditioned taste aversion (e.g. via postingestive nausea)by showing that workers avoid substrates harmful for theirsymbiotic fungus, but not for themselves. How robust these asso-ciations are is indicated by the marked long-term memory for plantunsuitability, which lasted up to 18 weeks. Recent studies on long-term memory in individual ants obtained values between 1 and 3days (Dreier et al. 2007; Josens et al. 2009; see also Johnson 1991and Johnson et al. 1994 for long-term memory and colonyresponses in seed-harvester ants). We conclude that the harvestingpattern observed in field colonies largely depends on the workers’foraging experience, which seems essential in a highly diverseenvironment where both leaf availability and quality varythroughout the year.

Acknowledgments

We thank the Editor and two anonymous referees for commentsthat helped improve the manuscript. This research was supportedby the German Academic Exchange Service (DAAD scholarshipgranted to N.S.), the Smithsonian Tropical Research Institute, andthe German Science Foundation (DFG), Sonderforschungsbereich567/TPC4 ‘Mechanisms of Interspecific Interactions of Organisms’.

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