The Journal Volume 45: 65-75 of Research JRl on the Lepidoptera ISSN 0022-4.S24 (print) THE LEPIDOPTERA RESEARCH FOUNDATION, 24 August 2012 ISSN 2I56-!')4.')7 (onunp)
Partitioning variation in duration of ant feeding bouts can offer insights into the palatability of insects: experiments on African fruit-feeding butterflies
Freerk Molleman’’^*, Ants Kaasik’, Melissa R. Whitaker^, James R. Carey‘S ' Institute of Ecology and Earth Sciences, University of Tartu, Vaneniuise 46, EE-51014 Tartu, Estonia * Department of Entomology, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA ’ Department of Evolution and Ecology, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA
Abstract. Quantification of chemical defence contributes to the study of animal signals, and to understanding trade-offs among defences and life history traits. Some tropical fruit-feeding butterfly species can be expected to have well-developed anti-predator defences because they are long-lived, are host-plant specialists, and/or have contrasting colourations that may be involved in mimicry' relationships. Yet, as a group they are often assumed to be palatable, even without supporting data. Palatability is a continuum that embraces within and between prey-species variation, and therefore, both among- and within-species variation must be documented. Palatability of nine species of fruit¬ feeding butterfly in Uganda was rated using a novel assay. One hundred and twenty-five butterflies were homogenized, their ground tissues suspended in sugar water and these suspensions offered as small dropleLs to individual ants in Petri dishes. The time ants spent feeding on the.se droplets was measured. Danaine butterflies were used as unpalatable references, and sugar solution as a palatable reference. Ants tended to eat in significandy shorter bouts from danaines compared to fruit¬ feeding species, and feeding bouts on pure sugar solution were longest Within fruit-feeding species, variation in the duration of ants’ feeding bouts was very substantial. There was also considerable variation among individual ants, such that large sample sizes would be needed to reliably distinguish palatability of different species of fruit-feeding butterflies. In explorative analy.se.s, at least three fniit- feeding butterfly species that were assumed palatable appeared to be chemically defended. These results suggest that, in contrast to common assumptions, some tropical fruit-feeding butterflies use unpalatability for defence, perhaps contributing to their long life spans in the wild.
Key words: fruit-feeding, tropical, Nymphalidae, mimicry, colour, chemical defence
Introduction having longevity records that exceed six months (Kelson, 2008; Molleman et aL, 2007, F. Molleman Quantification of chemical defence is important unpublished data), therefore they must have effective for understanding the evolution of signals to anti-predator strategies. Chemical defence in long- predators, investments in other types of defences, lived butterflies in the tropics is demonstrated by and life history. Tropical fruit-feeding butterflies several pollen-feeding butterfly species in the genus generally have long life spans, with many species Heliconius that have long active life spans similar to fruit-feeding species (Ehrlich & Gilbert, 1973; Engler- Chaouat Sc Gilbert, 2007; Pasteels Sc Gregoire, 1983; * Corresponding author. Institute of Ecology and Earth Sciences, Turner, 1971). Such association between chemical University of Tartu, Vanemuise 46, EE-51014 Tartu, Estonia. defence and long life spans in insects was proposed freerkmolU'man ©hotmail. com by Pasteels & Gregoire (1983), and butterflies that use more chemically defended host-plants live longer on average (Beck Sc Fiedler, 2009). However, in Received: 16 March 2012 Accej)ted: 5 June 2012 this latter multi-species comparison this effect was not statistically significant (Beck & Fiedler, 2009). One potential explanation for this could be that
Copyright: This work is licensed under the Creative Commons host-plant chemistry is a poor predictor of adult Attribution-NonCommercial-NoDerivs 3.0 Unported License. To palatability given varying levels of sequestration and view a copy of this license, visit http://creativecommons.org/ de novo synthesis, and thus chemical defence would licenses/by-nc-nd/3.0/ or send a letter to Creative Commons, 171 Second Street, Suite 300, San Francisco, California, 94105, be better measured using the butterflies themselves USA. (Beck & Fiedler, 2009). Furthermore, many fruit- 66 J. Res.I^pid.
feeding butterflies are host-plant specialists (but not Material and methods all, notable exceptions are grass feeding Satyrines and some Charaxes; e.g. DeVries, 1987; Larsen, 1991), Study site and subjects a trait associated with the sequestration of defensive chemicals (Nishida, 2002). Moreover, chemically This study -was conducted at the Makerere defended species often signal their unprofitability University Biological Field Station in Kibale Forest to predators with visual signals such as contrasting National Park, Western Uganda. The field station colour patterns (aposematism) and these signals borders selectively logged moist evergreen forest can then be mimicked by other species that are not at an altitude of around 1500 m (Chapman et necessarily defended. Such contrasting patterns are al., 2005). Palatability tests were conducted on found in many fruit-feeding butterfly species and nine fruit-feeding butterfly species (Lepidoptera: mimicry does occur within this guild, but evidence for Nymphalidae) illustrated in Fig. 1. Three species of the nature of it (Batesian versus Mfillerian, evasiveness danaines (Lepidoptera, Nymphalidae) were included versus palatability-mediated) is scarce. Therefore, it (AmaMrarawfim (Linnaeus, 1758), Tirumalapetiverana is likely that these colours function, at least in part, (Dovxbleday 1847), T. formosa (Godman 1880)) as to signal unpalatability to predators. examples of unpalatable butterflies (Jeffords et Despite these reasons to suspect chemical defence al, 1979) including evidence for African species in fruit-feeding butterflies, in the literature only a few but not the particular species used (Larsen, 1983, species of fruit-feeding butterfly have been shown to be 1992a, 2007). The fruit-feeding butterflies included chemically defended (e. g. Euphaedra cyparissa: Larsen, species with brightly coloured and contrasting wing 2007)—they are typically assumed to rely on evasive uppersides {E. eusemoides Grose-Smith & Kirby 1889, flight (Larsen, 1992b; van Someren &Jackson, 1959), E. alacris Hecq 1979), one species with deep blue/ crypsis, or eye-spots (Brakefield & Reitsma, 1991; Hill violet females and metallic green males {E. kakamega & Vaca, 2004; Marini-Filho & Martins, 2010) instead van Someren 1934), and species with cryptic wing of chemical defence. It should be tested whether fruit¬ patterns {Gnophodes chelysY^hvicms 1793, Kallimoides feeding butterflies that can putatively be classified as rumia Westwood 1850), as well as species that are evasive (e. g. Charaxes, Euphaedra) and/or cryptic (e. g. neither particularly cryptic nor clearly warningly Kallimoides, Gnophodes) also employ chemical defence in coloured {E. medon, E. harpalyce, Harma theobene the form of unpalatability. Finally, insight into within- Doubleday 1849, CharaxesfulvescensAxir'wWWu?, 1891). species variation in palatability is of interest. Various Larvae of most of these fruit-feeding butterflies are factors have been implicated in such variation, including considered cryptic. However, Euphaedra caterpillars age, sex, larval host-plant, and genetic differences in may be imprecise mimics of stinging slug-caterpillars defence strategy (Alonso-Mejia & Brower, 1994; Brower (Limacodidae), and contrasting colours are found et al, 1982; Eggenberger et ai, 1992; Eggenberger & in the gregarious caterpillars of E. kakamega (black Rowellrahier, 1992,1993; Holloway etai, 1993; Moranz with light yellow bands: Molleman & Hecq, 2005) & Brower, 1998; Saporito et ai, 2010). and to a lesser extent in those of E. eusemoides (green The present study used the duration of feeding with dark dorsal setae: Molleman, in press). The bouts of workers of one ant species on butterfly combination of warning colours and gregariousness suspensions as a measure of butterfly palatability. clearly indicates unpalatability (Sillen-Tullberg, Warningly coloured species were hypothesized to be 1988), and this is then usually transferred to the unpalatable, and chemical defence was hypothesized to adult stage as well (Pasteels & Gregoire, 1983). The play a role in an apparent mimicry relationship within host-plants from which the studied species were reared the genus Euphaedra: E. medon Thurau 1903 females in Kibale (Table 1) belong to families from which and E. harpalyceT?L\hoi 1929 (not closely related within various unpalatable or toxic chemicals are known (e.g. the genus). Mimicry in this group has been suspected Glaudino et al, 2009; Dongo et al, 2009; Krief et al, to be based on signalling of unprofitability based on 2006; Penders & Delaude, 1994; Webber & Woodrow, evasive flight (van Someren &Jackson, 1959), but strong 2009), except for the grass feeding G. chelys, while the evidence for such mimicry is lacking (Ruxton et al, host-plant of K. rumia is still unknown. 2004). The technique used to measure palatability was convenient in a field setting under tropical conditions, Experimental methods provided values for individual butterflies, was free of prejudice based on any visual signal, was (nearly) Butterflies were either reared from field-collected independent of odour-mediated attractiveness, and caterpillars (most fruit-feeding butterfly individuals subjects could be assayed over an extended period. except K. rumia) or were collected from the field as 45: 65-75, 2012 67
adults using sweep nets (danaines) or baited traps (all Table 1. Host-plant information for the assayed butterflies others). They were killed and the legs, wings, and head in Kibale National Park, Uganda. were removed before weighing. Each specimen was then ground up with three times its weight of boiled rainwater using a mortar and pestle. The resulting Butterfly Host-plant suspension was placed in a vial. In experiment I, a species genus family second solution was prepared in another vial that Asclepiadaceae, Danainae ? contained the same amount of water as was added to Euphorbiaceae the butterfly suspension, and a ten percent sucrose Euphaedra kakamega Aphania Sapindaceae solution was added to both vials so that both had a Euphaedra alacris Aphania Sapindaceae five percent sugar concentration. One droplet of each Euphaedra eusemoides Uvariopsis Annonaceae solution/suspension (one pair per Petri dish) was then Blighia, Aphania, Euphaedra harpalyce Sapindaceae placed two centimetres apart near the centre of a Petri Pancovia dish (droplets were named ‘butterfly’ and ‘sugar’). Euphaedra medon Paullinia Sapindaceae
In experiment II, the droplet of sugar solution was Harma theobene Lmdackeria Achariaceae omitted but the butterfly suspension was prepared Charaxes fulvescens Allaphylus Sapindaceae in the same way. Kallimoides rumia ? Three ant species that were common in the vicinity Gnophodes chelys Set aria Poaceae were tested in preliminary trials, but only Myrmicaria c.f. natalensis Smith 1858 (subfamily Myrmicinae) workers walked around quietly and fed from the solutions offered, while the others tended to sit at the Data analysis edge of the petri-dish without moving or ran about frantically. M. natalensis (Smith) (Hym: Formicidae) Each ant’s first choice of droplet (butterfly vs is a large, slow-moving, predaceous ant that forms sugar) in experiment I was recorded. Ants typically large nests of up to several thousand workers (Arnold, returned to the same droplet but some switches 1924) and is very common around Kibale National were observed as well, and these were expressed as Park. Worker ants from this species were collected proportions. Statistical analyses were performed from nearby ant trails and one ant was introduced using linear mixed models on ant feeding bout into each Petri dish with a soft forceps. They were durations in R (package lme4: Bates et al., 2011), left in the Petri dish for up to 90 minutes, and were residuals showing an adequate fit of the modelling replaced after 15 minutes of inactivity. Even though approach. 'Variation among individual ants and ants may show different behavior in their natural variation among butterfly individuals of the same context than when isolated in a Petri dish, workers of species were captured with random effects, with this species appeared to be reasonably at ease in our ants nested in butterfly individual. We attempted to set up. Similar arena trials for example in bio-activity correct for possible differences among experiment tests of larval sawfly haemolymphof (Muller et al, 2002) days by using weather data as covariates for all data, and for ranking the strength of interactions within and we used the duration of feeding bouts on sugar as and between species in the context of butterfly-ant daily references (for experiment I only). To determine mutualism (e.g. Ballmer & Pratt, 1991; Burghardt & whether a pooled analysis of data from experiments I Fiedler, 1996) have proven to be highly useful. and II was appropriate (the only difference between Observations were made by teams of two to five them being the presence/absence of the sugar local technicians. Each person could simultaneously droplet), we compared distributions of durations of observe up to six Petri dishes, each containing one two butterfly species that were well represented in pair of droplets (or one droplet in experiment II) and both data sets, and compared species effect estimates one ant, while one person recorded the data. Start between separate analyses of experiments I and II. and end times for each ant feeding bout were noted First, we tested whether fruit-feeding butterflies as in seconds. Local weather data were used to control a group could be distinguished from the references for any temperature effect. Experiment I was first (danaines and sugar solution) using one-tailed tests. supervised by EM (May-June 2007), was then carried Second, we tested whether there were significant on without supervision, and was later supervised differences among the fruit-feeding butterflies by MRW (July-August 2008). Experiment II was using a two-tailed test. Lastly, effect estimates for performed by local technicians without supervision butterfly species were calculated and compared to (September 2008-July 2009). the references using one-tailed tests. To compare 68 J. Res.Ixpid.
Figure 1: Photographs of butterfly species on which palatability tests were conducted, a. Euphaedra eusemoides female, b. Charaxes fulvescensma\e. c. E. a/acr/s female, d. E. harpa/yce female, e. E. medon female, f. E./ca/camega caterpillars, g. E./ca/camega female, h. E./ca/camega male. i. E.medon male. j. Gnophodes chelys female, k. Harma theobene male. I. Kallimoides rumia male.
durations of feeding bouts on butterfly suspension Results with those on sugar solution while using durations on sugar to correct for day effects, the feeding bout Experiment I yielded data from 57 butterflies (9 durations on sugar were subtracted from those on species), and experiment 11 from 68 butterflies (8 butterfly, and were then tested for equality with zero species) and the pooled data involved 663 feeding (data from experiment I only). ants. The first choice of ants was biased towards 45: 65-75, 2012 69
butterfly suspension for all species, including is up to 1.06 higher or lower than the average for danaines. Switching occurred mostly from sugar to the species, which is a lot because species estimates butterfly, except for danaines, E. kakamega, and H. range between 3.23 and 4.35. The magnitude of the theobene (Table 2). within- species variation can be illustrated further Feeding bout durations were seemingly gamma using E. harpalyce where the interval that includes distributed, ranging from a few seconds to about 10 about 95% of the individuals is 62 to 520 seconds. minutes. Log transformation produced a normally It was problematic to include butterfly age (freshly distributed response variable that was used for all emerged vs field collected) into the model, because of subsequent analyses and the graphical representation. unequal distribution among species and small sample Modelling log-transformed data using the normal sizes. Graphical representation did not suggest any distribution corresponds to the (untransformed) correlation between age (freshly eclosed vs field data being from the log-normal distribution. The collected) and palatability. For the species with the interval for the mean feeding bout duration within largest sample size {C. fulvescens) no significant age a species could, therefore, be estimated while taking effect was detected either. Moreover, no sex effect was into account the residual variation in the model. For found in our data, and including our weather data species that were well represented in both datasets, the did not improve our models. The variation among distribution of durations of feeding bouts on butterfly individual ants was also substantial with an SD for droplets were very similar (Kolmogorov-Smirnov the pooled data of 0.37 (Table 3). No effect of order 0=0.11, 0.42 for £. a/acraand 0 = 0.10,/>= 0.24 for C. number of feeding of individual ants was detected. fulvescens). Moreover, the estimates for species effects The analysis of feeding bout durations on pure were similar among the two experiments (Table 3). sugar showed that feeding bout durations differed Therefore, it wasjustified to combine the data sets for among days, and a similar pattern was detected this response variable. within the butterfly species such that there was a Within-species variation was extensive (Fig. correlation between feeding bout durations on 2). Because the random effects are assumed to be sugar and butterfly on particular days. However, normally distributed, we can analyze this variation in daily maximum temperature was not correlated with detail. For example, in the analysis of pooled data ant feeding bout duration on sugar. Sugar solution (Table 3), the standard deviation for the butterfly was only used in experiment 1, and for these data random effect was 0.54, meaning that for each species, correcting for such day effects by including durations individuals have a high probability of an average that on sugar as a co-factor improved the performance of
Figure 2. Palatability of butterfly species measured as duration of individual feeding bouts of ants (feedings) on butterfly suspensions (number of freshly emerged and field collected butterflies used). Box-plots represent averages among individual butterflies that are in turn based on varying numbers of feeding bouts with median, quartiles, and full range. The vertical dotted line represents the average duration of feeding bouts on sugar solution.
# fresh/field # feedings 0/11 116 Danainae -1 26/4 448 Charaxes fulvescens -1 1 4/0 73 Euphaedra kakamega 1 1- -1 n.... 13/5 287 Euphaedra alacris -J1 1 1 5/1 76 Euphaedra eusemoides -1 1 1- 3/6 189 Euphaedra harpalyce 1- --1 4/13 385 Euphaedra medon -1 1 6/7 162 Harma theobene -II 0/9 136 Kallimoides rumia -1 1 \- 6/2 121 Gnophodes chelys LL.
r X T X T X "1 1 2 3 4 5 6 7 mean log time (in seconds) 70 J. ResJ^pid.
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