Introduction

Ant-plant mutualisms are a common and widespread feature of both temperate and tropical ecosystems [1], [2]. These interactions generally involve plants providing food and/or shelter to ants in exchange for protection from herbivores and competitors [3]–[5]. Ant-plant mutualisms involve diverse taxa, particularly in the tropics, including at least 100 plant genera and 40 species of ants that engage in interactions from generalized and facultative to highly specialized and obligate [6], [7]. These conspicuous examples of indirect plant defense are model systems for understanding plant defense theory, food web structure, species coexistence, plant/animal coevolution, and the evolutionary stability of mutualisms [8]. However, ant-plant mutualisms have most often been studied from the perspective of one or both of the interacting partners, and we still have a limited understanding of the community context of ant-plant interactions—how other organisms (herbivores, predators, etc.) interact with plants and their ant defenders [9]–[14].

Numerous studies have demonstrated that ants are a highly effective defense mechanism that protects plants against a broad range of potential herbivores [8], [15]. However, no plant defense is insurmountable [16], [17], and there are examples of herbivores that are able to feed on ant-plants despite the presence of aggressive defenders [9], [18]. These herbivores may gain important benefits from using ant-occupied plants as hosts, including enemy-free space, reduced competition, and access to food sources produced by the plant as a reward for the ant mutualists. Furthermore, the leaf tissues of ant-defended plants often have lower levels of chemical defenses relative to unoccupied plants [19]–[21], and thus are likely to be more palatable to herbivores. Gaining access to ant-defended plants requires specialized traits that reduce ant attack rates or increase tolerance to attack, and past evidence has suggested a variety of possible traits may serve this function. Examples include toughened exocuticles [18], [22], shelter-building behaviors [9], and behavioral avoidance maneuvers [23], [24]. An improved understanding of these and other potential adaptations to ant-defense systems can provide insights into the evolutionary dynamics and maintenance of ant-plant-herbivore interactions, specifically, and the exploitation of mutualistic interactions in complex communities, in general.

It is likely that herbivores of ant-plants will exhibit adaptations similar to those typical of other organisms that interact closely with social insects, either as parasites, predators, or mutualists. Thus, the subject of herbivory on ant-plants can be informed by insights from the large body of research on the ecology and evolution of both ant social parasites [reviewed in 25,26] and mutualists (e.g. ant-tended aphids) [27], [28]. Although ant societies use complex behavioral and chemical cues for nestmate recognition and colony defense [29]–[31], an estimated 10,000 species of social parasites from 11 invertebrate orders have evolved specialized mechanisms that allow them to exploit ant colony resources [29], [32]. These parasites use a variety of mechanisms to infiltrate ant colonies or access resources, including chemical mimicry, acoustic mimicry, behavioral adaptations, specialized morphologies, and defensive compounds that deter, agitate, or confuse workers [18], [26], [32], [33]. In particular, many organisms gain access to ant colonies through a variety of strategies that circumvent the ants' ability to identify intruders, which is accomplished primarily through chemical recognition of cuticular hydrocarbons [25], [31], [34]. These strategies can be broadly characterized as chemical camouflage, which we define here as any chemical strategy that prevents detection or recognition (after Stevens and Merilaita [35], but see Dettner and Leipert [34] for a discussion of alternative definitions). There are many examples of ant social parasites that achieve chemical camouflage by mimicking the cuticular hydrocarbon profiles of their ant hosts; however this mode of avoiding detection can carry the high cost of host specificity, so that the parasite individual or species is confined to living with just one species of ant or even just a single colony [36]–[38]. Chemical camouflage may also be achieved by mimicking other components of the environment in which the interaction takes place. For example, recent evidence has shown that ant-tended aphids can avoid ant aggression and predation by mimicking the chemical profile of the host plant on which the species interact [28]. A third form of camouflage used by some ant social parasites is “chemical insignificance”, when infiltrating organisms have minimal levels of cuticular compounds that are essentially undetected by ants [25], [39]. Herbivores of ant-plants could avoid detection and attack through any of these different chemical camouflage strategies, used either alone or in combination with other strategies, such as chemical defense (e.g. via repellent sprays) or behavioral adaptations (e.g. furtive or defensive movements).

Perhaps the most well-studied example of ant-plant mutualism is the interaction between Pseudomyrmex spinicola Emery 1890 (Hymenoptera: Formicidae) ants and Neotropical bull-horn acacias (Vachellia collinsii (Saff. 1950) Seigler & Ebinger 2005, formerly Acacia collinsii, hereafter referred to as acacias; [40]). Acacia trees provide food for the ants in the form of extrafloral nectaries and Beltian bodies (nutrient-rich leaf structures fed to ant larvae), as well as domatia in the form of hollow thorns that are used as nest chambers by the ants [8], [18], [41]. In return, the ants defend host trees from herbivores and pathogens and reduce competition by clearing away encroaching vegetation [8], [18], [41]. Pseudomyrmex spinicola workers are highly aggressive and swarm from domatia to bite and sting almost any foreign object that comes in contact with their host tree [41]. Despite this conspicuous defense, a sap-feeding coreid (Piezogaster reclusus Brailovsky & Barrera 2000, Hemiptera: Coreidae) feeds primarily on ant-occupied acacias, undisturbed by resident ants (Fig. 1, personal observation, [42]). Both adult and juvenile P. reclusus feed primarily on the sap of young leaves, causing the young leaves to wilt and resulting in the loss of leaf tissue, Beltian bodies, and extra-floral nectaries. Yet, despite the potential direct costs of this damage to both the plants and the resident ants, there is no apparent recruitment of ant defenders nor increase in aggressive ant behavior when P. reclusus feeds or walks on the trees.

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Figure 1. Piezogaster reclusus feeding on a Vachellia collinsii tree occupied by Pseudomyrmex spinicola ants.

Photo by: André Kessler

https://doi.org/10.1371/journal.pone.0102604.g001

In this study, we tested three non-mutually exclusive hypotheses to explain how P. reclusus is able to avoid ant attack on acacia trees. First, we hypothesized that P. reclusus avoids ant attack through chemical camouflage (e.g. chemical mimicry or insignificance), as do many other invertebrates that infiltrate ant societies [28], [29], [34]. Second, we hypothesized that P. reclusus avoids ant attack through chemical defenses that are repellent to the ants. Many species of hemipterans, including members of the family Coreidae, defensively spray highly concentrated volatile organic compounds from glands on the metathorax [43], [44], some of which bear resemblance to ant-alarm pheromones and may have evolved in response to ant predation pressure [44]. Thus, it is possible that the defensive sprays of P. reclusus may also provide an important line of defense against ant attack. Third, we hypothesized that P. reclusus avoids ant attack through behavioral traits. Preliminary observations in this and one previous short-term study [42] indicated that in cases where the coreids are attacked, they often respond by leg-lifting, where an attacked leg is lifted into the air and a quick flick dislodges the ant. Other behaviors may also be important in reducing detection by ants, such as walking softly to avoid movement of the branch that would cause ant alarm.

To test these hypotheses, we first conducted a series of field experiments designed to isolate the role of cuticular chemistry, glandular spray, and behavior in allowing P. reclusus to avoid ant attack. This involved examining the responses of P. spinicola ants to: 1) compounds extracted from P. reclusus cuticles or metathoracic glands, or 2) P. reclusus or other herbivores that do not feed on acacia trees, treated in various ways to isolate the effects of cuticle chemistry, glandular sprays, and behavior. Next, to further test the hypothesis of chemical camouflage, and to distinguish between different potential camouflage strategies (specifically mimicry versus chemical insignificance), we examined the host-specificity of P. reclusus on acacia trees occupied by different colonies of P. spinicola. Because effective chemical mimicry of ants or host plants would require P. reclusus to have cuticular hydrocarbon profiles that are colony-specific, we predicted that if the coreids avoided attack through chemical mimicry they would experience increased levels of ant aggression when moved to new host trees.

Materials and Methods

Results

Ant responses to extracted chemical compounds

In the “cuticular compounds” experiment (Exp. 1a), cotton swabs treated with extracted cuticular compounds were attacked in 16% of encounters, significantly less often than lanolin-only controls, which were attacked in 33% of encounters (GLMM; Χ² = 12.73, df = 1, p = 0.00036; Fig. 2A). In the “glandular compounds” experiment (Exp. 1b), cotton swabs treated with glandular compounds were attacked in 3% of encounters, significantly less often than controls, which were attacked in 77% of encounters (GLMM; Χ² = 51.51, df = 1, p<0.0001; Fig. 2B).

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Figure 2. Response of Pseudomyrmex spinicola ants to isolated compounds from Piezogaster reclusus.

Bars show the mean proportion (± SE) of encounters in which Pseudomyrmex spinicola ants attacked cotton swabs that were treated with Piezogaster reclusus (A) cuticular compounds or (B) glandular compounds in comparison to controls. Letters indicate significant differences from binomial GLMMs that included ant colony as a random effect.

https://doi.org/10.1371/journal.pone.0102604.g002

Ant responses to coreids and other herbivores

In the “cuticular washes” experiment (Exp. 2a), water-washed coreids were attacked in 24% of encounters, MeOH-washed coreids were attacked in 27% of encounters, and hexane-washed coreids were attacked in 38% of encounters. There was an overall effect of wash treatment on the proportion of ant attacks (GLMM; Χ² = 16.20, df = 2, p = 0.00030). Post-hoc comparisons among treatments showed that hexane-washed coreids were attacked significantly more often than water-washed (p = 0.00053) or methanol-washed (p = 0.0072) coreids (Fig. 3A). There was no significant difference between methanol-washed and water-washed coreids.

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Figure 3. Response of Pseudomyrmex spinicola ants to Piezogaster reclusus and other herbivores.

Bars show the mean proportion (± SE) of encounters in which Pseudomyrmex spinicola ants attacked: (A) dead individuals of Piezogaster reclusus that were washed in water, methanol, or hexane to remove cuticular compounds, or (B) individuals of Dysdercus sp. that were treated with lanolin-extracted Piezogaster reclusus cuticular compounds or lanolin only as a control. Letters indicate significant differences from binomial GLMMs that included ant colony as a random effect.

https://doi.org/10.1371/journal.pone.0102604.g003

In the “glandular spray and behavior” experiment (Exp. 2b), we conducted five replicate trials with 140 total ant encounters, and observed only two occurrences of an ant attacking P. reclusus, both on the same unrestrained individual with open glands. Other restrained herbivores were attacked, on average, in 39% of encounters, which was significantly more often than coreids that were restrained with their glands sealed (GLMM; Χ² = 23.17, df = 1, p<0.0001).

In the “cuticular compound transfer” experiment (Exp. 2c), individuals of Dysdercus sp. treated with lanolin containing coreid cuticular compounds were attacked in 33% of encounters, significantly less often than those treated with lanolin only, which were attacked in 37% of encounters (GLMM; Χ² = 4.60, df = 1, p = 0.032; Fig. 3B).

Host specificity of coreids (P. reclusus)

In the “host colony specificity” experiment (Exp.3), coreids translocated to new trees occupied by different P. spinicola ant colonies were attacked in 54% of encounters, significantly more often than coreids replaced on their tree of origin, which were attacked in 20% of encounters (GLM; z = 2.39, df = 8, p = 0.017; Fig. 4).

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Figure 4. Response of Pseudomyrmex spinicola ants to replaced or translocated individuals of Piezogaster reclusus.

Bars show the mean proportion (± SE) of encounters in which Piezogaster reclusus was attacked when replaced on the host tree from which it was collected or translocated to a new tree with a different ant colony. Letters indicate significant differences from a binomial GLM.

https://doi.org/10.1371/journal.pone.0102604.g004

Discussion

In the classic example of ant-plant mutualism between Neotropical bull-horn acacias and Pseudomymex ants, a number of herbivores have been described that feed on acacias undisturbed by resident ants [9], [18], [23], [54]. However, the mechanisms by which these organisms are able to circumvent the highly specialized defense system of the ants are often poorly understood. Our results show that non-polar compounds present on the cuticles of a coreid bug, P. reclusus, provide protection against attack from P. spinicola ants. Other mechanisms of avoiding attack, such as specialized behaviors or deterrent compounds sprayed from the coreid metathoracic gland, may also play a role in reducing attack, but the coreids' ability to feed among ants seems to persist even when these traits are experimentally removed. Furthermore, our experiments show that P. reclusus bugs experience increased levels of attack when transferred among ant colonies, suggesting that the protection provided by coreid cuticular chemistry is colony-specific. Although many questions remain about the specific chemical cues involved and their mode of action, our results provide the most support for the hypothesis that chemical camouflage, in general, and chemical mimicry, in particular, of either ant or plant cuticular hydrocarbons is a key mechanism that allows P. reclusus to feed on acacia trees while avoiding attack.

Results from our field experiments with extracted cuticular compounds, coreids receiving various wash treatments, and other insects with coreid cuticular compounds transferred to their cuticles (Fig 2A, Fig. 3A, Fig. 3B, respectively) show that the compounds present on coreid cuticles provide protection against ant attack and are consistent with the hypothesis that these compounds mimic either compounds present on ant cuticles or on the plant surface. Lanolin extracts of non-polar cuticular compounds from live coreids reduced ant attack rates when presented on cotton swabs (Fig. 2A) and also provided some protection for other herbivorous insects (Dysdercus sp.) when transferred to their cuticle surfaces (Fig. 3B). Furthermore, coreids whose cuticular chemistry had been altered/removed via hexane washes were attacked significantly more than those washed in MeOH or water (Fig. 3A). Although we expected that MeOH washes would also remove some cuticular hydrocarbons and increase attack rates relative to water-washed insects, there was only a slight, non-significant trend towards increased attack on MeOH-washed relative to water-washed insects. It is likely that the methanol was less efficient than hexane at removing the relevant hydrocarbons from the cuticle surface, suggesting highly non-polar cuticular hydrocarbons as the putative mimicry signal. Thereby P. reclusus cuticular chemistry could mimic either ant or plant cuticular hydrocarbon composition or both. To our knowledge the cuticular hydrocarbons present on P. spinicola have not been identified, but the profiles of closely related species of Pseudomyrmex are dominated by highly non-polar mono- and dimethyl-alkanes varying in chain length from C27 to C37 [55]. Even less is known about acacia surface chemistry, which equally likely could provide cues either for the P. reclusus bugs to mimic the host plant and therefore be undetected by ants, or for ants and bugs to express similar cuticular chemistry as a result of similar dietary intake [56], [57].

Our experiments also partially supported our second hypothesis, that defensive compounds produced in the coreid methathoracic glands reduce the level of ant attack. Glandular compounds on cotton swabs resulted in a nearly 30-fold decrease in the proportion of encounters that were attacks (Fig. 1B), providing strong evidence that these compounds are deterrent to ants. However, our experiments with coreids that had their glands experimentally closed indicated that coreids are still rarely attacked by ants even without their ability to use glandular sprays as a defense. The fact that ants were active in the experiment (with 140 total encounters with coreids) and were aggressive towards other herbivores suggests that glandular defensive spray is non-essential in avoiding attack by ants on the host tree. Furthermore, our results showing that sealing glands has no effect on ant attack rates were similar to results found in a preliminary study [42]. However, these results do not preclude the possibility that glandular sprays may be used as an important line of defense when coreids are attacked. Interestingly, in our experiment with glandular compounds on cotton swabs, we observed a strong tendency for ants that encountered the treated swabs to avoid the stimulus, usually backing away and changing direction, rather than passing by and continuing in the same direction as they often did when the stimulus involved a coreid or extracted cuticular compounds. This behavior suggests that ants were in fact detecting the glandular compounds, but were repelled by the odor rather than initiating typical defense behaviors. Other species in the family Coreidae are known to use volatile glandular compounds in defense [44], and these chemicals may have a generalized deterrent function against ants as well as other predators or natural enemies.

Our third hypothesis, that behaviors such as leg-lifting or walking softly can also play a role in protecting coreids against ant attack, was not supported in this study. Our treatment of restraining coreids by gluing them to the plant clearly prevented normal behaviors, but led to no detectable increase in ant attack. However, when coreids were free to move around on the plant, we did observe the leg-lifting behavior as noted in an earlier study [42]. Our results do not preclude the possible importance of these and other behaviors on occasions when ants do attack coreids, but suggest that, like the glandular spray, behavior is not the primary mechanism through which coreids are able to persist on acacia trees.

Our final set of experiments was designed to further test the hypothesis of chemical mimicry by determining the host specificity of coreids to different colonies of P. spinicola. Because cuticular hydrocarbons are used by ants in recognition at the nestmate level, we predicted that if coreids do use chemical mimicry to avoid attack, individuals would be specialized with respect to ant colony. We did find that coreids were attacked more often when translocated to new colonies of P. spinicola (Fig. 4). Other explanations for how cuticular hydrocarbon chemistry may be involved in reducing attack, such as the “chemical insignificance” hypothesis [25], [39] or the possibility that coreid cuticular compounds are somehow deterrent or confusing to the ants, would predict no change in ant aggression when coreid bugs are moved from home ant colonies to foreign ant colonies. Thus, these results provide important additional support for the hypothesis of chemical mimicry and also have implications for understanding the constraints imposed by this strategy in terms of host plant use and dispersal by P. reclusus.

It is unclear whether coreids synthesize cuticular hydrocarbons or acquire them from the host ant colony or host plant. Because the entire life cycle of P. reclusus is associated with acacia trees, including egg and nymph stages (personal observation), one possibility is that host colony odors are acquired by the coreids early in development and single individuals spend the majority of their lives on a single tree. Reproductive individuals may later disperse to trees occupied by different colonies, and although our results suggest host-switching may be initially costly in terms of increased attack rates, coreids may acquire host colony or host plant odors over time and eventually feed on the new tree undisturbed. Even if adult coreids do not acquire new host colony odors, they may be able to avoid ant attack through a combination of chemical deterrence and behavior for long enough to mate and lay eggs on a new tree. Future work examining the composition and changes in cuticular hydrocarbon profiles during P. reclusus development and how these changes relate to the dispersal ability of the coreids among ant colonies and host trees will help to identify the potential costs involved in chemical mimicry and its effects on P. reclusus demography.

Our study provides the first description of the mechanisms by which a coreid (P. reclusus) is able to overcome the ant defenses of the Neotropical bull-horn acacias (Vachellia collinsii), and our results are consistent with the hypothesis that coreids use chemical mimicry to avoid aggression from the ants. Future work in this system should seek to: 1) characterize the cuticular hydrocarbon profiles of ants, coreids, and their common host plants, 2) determine whether coreid cuticular hydrocarbons are synthesized de novo or acquired from ant colonies or host plants, and 3) further examine the dispersal of coreids among host trees and the variation in ant-acacia-coreid interactions in space and time. The ant-acacia interaction is a textbook example of mutualism that is considered to be highly specialized and tightly coevolved [8], [41]. However, herbivores are able to overcome the ability of ants to effectively defend their host trees, and exploitation of the chemical recognition system of the ants is one important way through which this can occur. An improved understanding of the mechanisms and outcomes of these interactions can provide new insights into the effectiveness of biotic defense against herbivory and the evolution and maintenance of mutualistic interactions.

Acknowledgments

We gratefully acknowledge the staff of Palo Verde National Park and the Organization for Tropical Studies for providing logistical support for this study. Permits for research in Costa Rica were obtained from the Ministerio de Ambiente y Energía (MINAE, No. 01758, Resolución No. 048-2008-SINAC) and we are grateful to Edwin Lynch for assistance with Spanish translation of the permit application. Harry Brailovsky of the Instituto de Biología, Universidad Nacional Autónoma de México provided assistance with identification of P. reclusus. The manuscript was greatly improved by discussion and comments from Bruce E. Lyon, Robert Montgomerie, Erika Deinert, M. Deane Bowers, Amy Churchill, Tobin Hammer, and the Plant-Animal Interactions discussion group at the University of Colorado.