Oviposition tests of ant preference in a myrmecophilous butterfly

Butterflies in the family Lycaenidae that have obligate associations with ants frequently exhibit ant‐dependent egg laying behaviour. In a series of field and laboratory choice tests, we assessed oviposition preference of the Australian lycaenid Jalmenus evagoras in response to different species and populations of ants. Females discriminated between attendant and nonattendant ant species, between attendant ant species, and to some extent, between populations of a single ant species. When preferences were found, ovipositing butterflies preferred their locally predominant attendant ant species and geographically proximate attendant ant populations. A reciprocal choice test using adults from a generation of butterflies reared in the absence of ants indicated a genetic component to oviposition preference. Individual females were flexible with respect to oviposition site choice, often ovipositing on more than one treatment during a trial. Preferences arose from a hierarchical ranking of ant treatments. These results are discussed in terms of local adaptation and its possible significance in the diversification of ant‐associated lycaenids.     

The distribution and density of a lycaenid butterfly in relation to Lasius ants

Summary Larvae and pupae of lycaenid butteflies are often associated with ants: this is usually a mutualism in which ants guard the lycaenids from natural enemies, and the lycaenid larvae and pupae provide sugars and amino acids for the ants. A possible consequence of the interaction is spatially correlated ant and lycaenid distributions, but the phenomenon is poorly documented. We examined the lycaenid Plebejus argus, which is tended by Lasius ants. Within habitat patches, P. argus eggs, larvae and pupae were all spatially associated with Lasius. On a larger scale, the densities of butterflies in different habitat patches and populations, and whether the butterfly was present or not, were correlated with Lasius ant densities. The association of P. argus with Lasius ants is consistent among populations, and occurs at several spatial scales. Other aspects of the ecology of P. argus are more variable.     

A single lycaenid caterpillar gets an ant‐constructed shelter and uninterrupted ant attendance

Ant‐lycaenid associations range from mutualism to parasitism and the caterpillars of some species of lycaenids are reported to enter ant nests for shelter, diapause, or pupation. The present study aimed to examine the nature of the association between Euchrysops cnejus (Fabricius) (Lepidoptera: Lycaenidae) and Camponotus compressus (Fabricius) (Hymenoptera: Formicidae) worker ants on the extrafloral nectary‐bearing cowpea plant, Vigna unguiculata (L.) Walp. (Fabaceae). The abundance patterns of the ants and the lycaenid caterpillars together with the spatial patrolling patterns of the ants on the plants revealed that ant abundance increased with the occurrence of the lycaenid caterpillars and the ants preferred the lycaenids over the extrafloral nectar. Camponotus compressus worker ants constructed a shelter at the cowpea plant base after interaction with one or more lycaenid caterpillar(s) and tended the caterpillars and pupae till the emergence of the butterfly. The ant‐constructed shelters (ACSs) inhabited by the minor caste workers (13 ± 1.3 ants per ACS), were utilized by the caterpillars to undergo pupation. The ants confined their activities predominantly to tending the pod‐feeding caterpillars and the solitary pupa within each ACS. It appears that the behavior of the tending worker ants is modulated by the lycaenid vulnerable stages.     

Timing of butterfly parasitization of a plant–ant–scale symbiosis

In the Southeast Asian tropics, Arhopala lycaenid butterflies feed on Macaranga ant-plants inhabited by Crematogaster (subgenus Decacrema) ants tending Coccus-scale insects. A recent phylogenetic study showed that (1) the plants and ants have been codiversifying for the past 20–16 million years (Myr), and that (2) the tripartite symbiosis was formed 9–7 Myr ago, when the scale insects became involved in the plant–ant mutualism. To determine when the lycaenids first parasitized the Macaranga tripartite symbiosis, we constructed a molecular phylogeny of the lycaenids that feed on Macaranga by using mitochondrial and nuclear DNA sequence data and estimated their divergence times based on the cytochrome oxidase I molecular clock. The minimum age of the lycaenids was estimated by the time-calibrated phylogeny to be 2.05 Myr, about one-tenth the age of the plant–ant association, suggesting that the lycaenids are latecomers that associated themselves with the pre-existing symbiosis of plant, ant, and scale insects.     

Aphytophagy in butterflies: its relationship to myrmecophily

The regular or obligate aphytophagy of certain lycaenid butterflies (Lepidoptera) is discussed within the framework of the most recent general classification of the family. A summary survey of all Lycaenidae known to be aphytophagous is presented, together with a brief account of cannibalism and other opportunistic aphytophagy exhibited by normally phytophagous butterflies. The range of food sources (plants, animals, excretions and regurgitations) exploited by lycaenids is reviewed with emphasis falling on the ecology of myrmecophilous early stages and the significance of their ant-related adaptations. Adult feeding and oviposition behaviour reveal further associations with ants. Specificity of lycaenid/ant relationships and the possible biological effects of aphytophagy on the Lycaenidae are discussed. Finally, speculations concerning the evolution of aphytophagy by these butterflies are critically presented.     

Identifying potential evolutionary relationships within a facultative lycaenid-ant system： Ant association, oviposition, and butterfly-ant conflict

Facultative associations are commonly encountered between ants and lycaenids, although the nature and patterns of associations are typically unclear. This study investigated a facultative symbiosis involving the lycaenid Theclinesthes albocincta （Lycaenidae）, its host plant Adriana quadripartita and Australian native ants. Ants in the genera Ochetellus and Iridomyrmex were most frequently found in association with T. albocincta larvae, although Iridomyrmex ants were found in much lower abundance than were ants in Ochetellus. The abundances of Ochetellus and Iridomyrmex were highly correlated with larval abundance, but not egg abundance. Observations and experiments recorded oviposi- tion on male inflorescences on more than 95% of occasions, but oviposition was not greater on inflorescences with ants present. Behavioral assays showed that Iridomyrmex ants were aggressive towards female butterflies on significantly more occasions than were Ochetellus ants. These findings indicate potential evolutionary relationships between T. albocincta and two genera of ants that were abundant within the habitat.     

Mutualistic ants and parasitoid communities associated with a facultative myrmecophilous lycaenid,Arhopala japonica,and the effects of ant attendance on the avoidance of parasitism

Herbivorous insects have evolved various defensive strategies to avoid their primary enemies, parasitoids. Many species of Lycaenidae (Lepidoptera) have food‐for‐protection mutualism with ants in their larval stages, where larvae produce nectar for ants and in return ants exclude parasitoids as well as predators. Myrmecophilous relationships are divided into two categories, obligate and facultative, by degrees of myrmecophily. Although parasitoids attacking obligate lycaenids always encounter lycaenid‐specific ant species, parasitoids that use facultative lycaenids are likely to encounter diverse ant species showing various defense systems. However, we know little about the parasitoid community of facultative lycaenid larvae. In this study, we investigated the mutualistic ant and parasitoid communities of a facultative myrmecophilous species, Arhopala japonica, in seven localities in Japan. The present field observation newly recorded four ant species attending A. japonica larvae, and combined with the previous data, the number of attending ant species reached 16, which is nearly the maximum number of reported attending ant species among myrmecophilous lycaenids. However, the present study revealed that almost all parasitized A. japonica larvae were attacked by a single braconid species, Cotesia sp. near inducta. We also assessed the efficiency of facultative ant defense against the parasitoid in the laboratory and revealed that oviposition by Cotesia sp. near inducta females was almost completely hindered when A. japonica larvae were attended by ants. This suggests that the dominant parasitoid does not have effective traits to overcome defensive behavior of ants and that the female wasps oviposit mainly in A. japonica larvae without intensive attendance.     

Evolution of influence: signaling in a lycaenid-ant interaction

Some phytophagous insects gain defense from natural enemies by associating with otherwise potentially harmful top predators. Many lycaenid butterfly caterpillars are involved in such interactions with ants: larvae provide carbohydrate rewards from the dorsal nectary organ (DNO) to associated ants in return for protection from natural enemies. The stability of these interactions involves signals that identify the lycaenid caterpillar as a mutualist. However, larvae of some lycaenid species, such as Lycaena xanthoides, are found in close association with ants but do not possess the reward producing DNO. Evaluating the relationship in a phylogenetic framework, we show that the association between L. xanthoides and ants likely evolved from a non-ant-associated ancestor. Behavioral trials also show that L. xanthoides larvae are capable of influencing ant behavior to increase ant tending when faced with a simulated predator attack, without providing DNO-derived rewards to ant associates. These results demonstrate that the DNO is not necessary to maintain associations between lycaenid larvae and ants. Third-party interactions may affect the evolution of mutualisms and consideration of underlying evolutionary history is necessary to understand contemporary species associations.     

The influence of ants on patterns of colonization and establishment within a set of coexisting lycaenid butterflies in a south-east Asian tropical rain forest

In Peninsular Malaysia ten species of lycaenid butterflies use leaf flushes or inflorescences of the legume tree Saraca thaipingensis as larval hostplant. Resource partitioning among these species is regulated by a complex mixture of patterns of interaction with ants. Females of obligately myrmecophilous species lay their eggs exclusively on trees colonized by their specific host ants. On trees colonized by weaver ants, only specialist mutualists adapted to these territorial ants are able to survive, while larvae of other species are killed. The formicine ant Cladomyrma petalae, which inhabits hollow twigs of the myrmecophytic hostplant, likewise precludes oviposition by female butterflies. Lycaenid larvae confronted with this ant species never survive, but one concealed feeding species (Jamides caeruleus) escapes removal due to the cryptic life-habits of the larvae. Two facultative myrmecophiles associate in a mutualistic way with a wide and largely overlapping range of ant genera which forage at the extrafloral nectaries of leaf flushes. One species (Cheritra freja) is not myrmecophilous, but is tolerated by all but the most territorial ants. Ant-dependent hostplant selection and egg-clustering characterize the obligate mutualists, whereas facultative myrmecophiles and the non-myrmecophile distribute their eggs singly over appropriate hostplants. Signals mediating caterpillar-ant communication are highly specialized in one obligate myrmecophile (Drupadia theda), but rather unspecific in four other species tested. Altogether our observations indicate that colonization and establishment of lycaenid butterflies on S. thaipingensis trees are governed by specializations as well as opportunistic use of resources (ants and hostplant parts). Therefore, the diversity of this species assemblage is maintained by deterministic as well as stochastic factors.     

Association between the African lycaenid,Anthene usamba,and an obligate acacia ant,Crematogaster mimosae

The African lycaenid butterfly, Anthene usamba, is an obligate myrmecophile of the acacia ant, Crematogaster mimosae. Female butterflies use the presence of C. mimosae as an oviposition cue. The eggs are laid on the foliage and young branches of the host plant, Acacia drepanolobium. Larvae shelter in the swollen thorns (domatia) of the host tree, where they live in close association with the acacia ants, and each larva occupies a domatium singly. Anthene usamba are tended by ants that feed from the dorsal nectary organ at regular intervals. Larvae also possess tentacle organs flanking the dorsal nectary organ and appear to signal to ants by everting these structures. Larvae were observed to spend most of their time within the domatia. Stable isotope analysis of matched host plant–ant–butterfly samples revealed that Anthene usamba are δ¹⁵N enriched relative to the ants with which they associate. These data, based on the increase in δ¹⁵N through trophic levels, indicate that the caterpillars of these butterflies are aphytophagous and either exploit the ant brood of C. mimosae within the domatia, or are fed mouth to mouth by adult workers via trophallaxis. This is the first documented case of aphytophagy in African Anthene. Pupation occurs inside the domatium and the imago emerges and departs via the hole chewed by the larva. The adult females remain closely associated with their natal patch of trees, whereas males disperse more widely across the acacia savannah. Females prefer to oviposit on trees with the specific host ant, C. mimosae, an aggressive obligate mutualist, and avoid neighbouring trees with other ant species. Adult butterflies are active during most months of the year, and there are at least two to three generations each year. Observations made over a 5‐year period indicate that a number of different lycaenid species utilize ant‐acacias in East Africa, and these observations are summarized, together with comparisons from the literature. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013 , 109 , 302–312.     

A comparative analysis of morphological and ecological characters of European aphids and lycaenids in relation to ant attendance

Ants are a major environmental factor for many insect species. For example, aphids and lycaenids have evolved an array of associations with ants ranging from obligate myrmecophily to the avoidance of contact. Here we (1) analyze the predictive power of different ecological and morphological traits for explaining the strength of the association between ants and aphids/lycaenids and (2) contrast different taxonomic levels with respect to the variance explained by ant attendance. Data come from a literature survey including 112 species of aphids and 103 species of lycaenids from Europe. For aphids, feeding on woody plant parts is positively associated with ant attendance, while a high degree of mobility, feeding in isolation, and the possession of wings in the adult stage are negatively associated with ant attendance. In lycaenids, feeding on inflorescences and feeding on Fabaceae host plants is closely associated with ant attendance, while living in forests bears a smaller likelihood to establish mutualistic relationships. Body size always appeared to be a poor predictor for the degree of ant attendance. Overall, in both insect groups less than 10% of the variation in the ecological traits recorded is explained by the different modes of ant association. When decomposing the variance in traits explained by ant attendance at different taxonomic levels, aphids and lycaenids show contrasting results. In aphids, most variance in the degree of ant attendance is explained at the subfamily level and least at the species level. The opposite is true for lycaenids, where most variance is explained at the lowest taxonomic level. Possible mechanisms explaining these different patterns of associations with ants are suggested.     

Ants benefit from attending facultatively myrmecophilous Lycaenidae caterpillars: evidence from a survival study

Workers of three ant species (Lasius niger, Lasius flavus, Myrmica rubra) were caged in the laboratory together with caterpillars and pupae of five species of lycaenid butterflies. Mortality of ants was 3–5 times higher when the ants were confined with larvae lacking a dorsal nectar organ (Lycaena phlaeas, Lycaena tityrus) rather than with caterpillars which possess a nectar gland (Aricia agestis, Polyommatus bellargus, P. icarus). For all five species, ant survival was always lower at the pupal stage (where a nectar organ is always absent) than at the caterpillar stage and was largely equivalent for the butterfly species tested. The experimental data confirm earlier estimates that ants can derive nutritive benefits from tending facultatively myrmecophilous lycaenid caterpillars, even though these caterpillars produce nectarlike secretions at low rates.     

Of crazy-ants and Curetinae: are Curetis butterflies tended by ants?

The larvae of the lycaenid subfamily Curetinae have never been reported to be associated with ants. Observations on Curetis regula Evans from Brunei are presented which show that this species may be tended by ants both as larvae and adults. The observations are discussed in relation to a recent review on lycaenid/ant associations, u is suggested that the Curetinae will be found to be associated with ants when more species have been reared, on evidence of the larval tentacle organs and apparent ‘pore cupolas’, both of which are ant adaptations. More studies are needed on Curetis biology and larval morphology to resolve the relationships of this enigmatic genus within the Lycaenidae.     

Behavioral strategy of a lycaenid (Lepidoptera) caterpillar against aggressive ants in a Brazilian savanna

Myrmecophily is widespread in lycaenid butterflies, in which ants receive food resources and, in turn, protect caterpillars against natural enemies. This interaction ranges from obligate myrmecophily, in which immatures are invariably associated with ants and are dependent on ants for survival, and facultative myrmecophily, in which larvae are not dependent on ants for survival, but the presence of the latter may increase larvae survival. Lycaenids also include non-myrmecophilous butterflies, which do not have positive associations with ants and have developed strategies to avoid being attacked or preyed upon by them. In this study, we examined the relationship between the lycaenid Michaelus ira and two ant species associated with Distictella elongata (Bignoniaceae). This plant has extrafloral nectaries and is patrolled by Camponotus crassus and Ectatomma tuberculatum. Morphological analyses revealed that M. ira larvae have ant organs, such as dorsal nectary organs and perforated cupolas, structures associated with myrmecophily. We performed larval exposure experiments in the field, predicting that, in the absence of myrmecophily, the butterfly larva would present strategies to avoid ant attack. Results showed that larvae were attacked by both ant species. To escape ant molestation, larvae lived and fed inside silk-sealed D. elongata flower buds. We concluded that the M. ira bud-sheltering behavior was a defensive strategy against these ant species, while the dorsal nectary organs were apparently nonfunctional. Nonetheless, myrmecophily, in general, cannot be excluded in M. ira since relationships with other ant species may exist.     

Australian parasitic Ogyris butterflies: east–west divergence of highly‐specialized relicts

Not all butterflies are innocuous plant‐feeders. A small number of taxa in the family Lycaenidae have graduated from mutualistic partnerships with ants to predatory or parasitic associations. These highly‐specialized life histories, involving butterfly larvae living inside ant colonies, are often associated with rarity and vulnerability to extinction. In the present study, we examined the evolutionary relationships of a poorly‐known group of seven taxa herein referred to as the idmo‐group within the Australian lycaenid genus Ogyris. The idmo‐group has a relictual distribution across southern Australia and includes taxa with highly‐specialized phytophagous and myrmecophagous life histories. A phylogeny based on mitochondrial DNA (cytochrome oxidase I and cytochrome b] and the nuclear DNA locus elongation factor 1α (EF1α), generally agrees with current taxonomy and supports the recent elevation of endangered taxon Ogyris halmaturia to full species status. The transition to myrmecophagy was dated to the mid‐Miocene (approximately 16 Mya), when southern Australia experienced a humid climate and extensive mesic biome. The arid Nullarbor Plain, a major biogeographical feature of central southern Australia, divides the remnants of this mesic biome into south‐eastern and south‐western isolates. Late‐Miocene to Pliocene divergence estimates for polytypic Ogyris species across the Nullarbor were older than estimates made for similarly distributed birds, butterflies, mammals, and reptiles, which mostly date to the Pleistocene. The concept of highly‐specialized life histories as evolutionary dead‐end strategies is well exemplified by the idmo‐group. Data compiled on the known extant subpopulations for idmo‐group taxa show that all of these extraordinary butterflies are scarce and several face imminent threat of extinction. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 473–484.     

Absence of cuticular alkenes allows lycaenid larvae to avoid predation by Formica japonica ants

Chemical mimicry and camouflage based on cuticular hydrocarbons (CHCs) are adaptive strategies that are frequently observed in myrmecophilous insects. The larvae of several lycaenid butterfly species that exhibit obligate associations with specific ant species have been reported to use chemical mimicry. However, little is known about the strategies used by the larvae of species that have facultative associations with multiple ant species. We attempted to reveal the effects of larval CHC profiles on interactions with Formica japonica workers, using three lycaenid species, two facultative ant‐associated (Lycaeides argyrognomon and Zizeeria maha) and one non‐ant‐associated (Lycaena phlaeas), which commonly possess n‐alkanes as the major CHCs. In field bioassays, the lycaenid larvae were attacked by ant workers less often than larvae of Papilio polytes (Papilionidae), the CHCs of which were rich in 7‐alkenes. Treating the lycaenid larvae with 7‐heptacosene and 9‐heptacosene significantly activated ant aggression (biting), whereas treating them with n‐heptacosane, n‐octacosane and 13‐methylheptacosane had little effect. Furthermore, larvae of Pieris rapae (Pieridae), possessing n‐alkanes as the dominant CHCs, suffered an intermediate level of ant biting between the lycaenid and Pa. polytes larvae. However, treatments of the P. rapae larvae with 7‐heptacosene and 9‐heptacosene significantly affected the frequency of ant biting. These findings suggest that the absence of alkenes in larval CHC profiles is an effective means of circumventing predation by ants and allows lycaenid larvae to inhabit the foraging territory of predaceous ants, at least to some extent.     

The acoustic repertoire of lycaenid butterfly larvae

Sounds and vibrations play vital roles in intra- and inter-specific communication of many insect taxa, for sexual display, defence and social recruitment. In Lepidoptera, sound production occurs in larvae, pupae and adults and has evolved in response to selection of sexual or defensive traits. About 75% of the 6000 estimated lycaenid butterflies are associated with ants (termed “myrmecophilous species”) and many species produce acoustic emissions during pre-imaginal development. It was initially believed that these acoustic emissions were only produced by myrmecophilous species, but later studies showed that the ability to produce sounds may be universal among this butterfly family. The acoustic repertoire of the late-instar larvae of 12 lycaenid species (Polyommatinae and Lycaeninae), showing different degrees of interaction with ants, was analysed by investigating 12 acoustic parameters measured on the call fundamental unit (pulse). All samples produced species-specific calls whose spectra were characterized by harmonic frequency components. The inter-specific call diversity better reflects the level of association with ants than the taxonomic relationships between species. Our results support the hypothesis that the ability to emit acoustic signals is widespread in lycaenids, and that these emissions play a role in myrmecophilous interactions.     

Aggregation as a cost-reducing strategy for lycaenid larvae

If a mutualistic relationship entails providing services ata cost, selection will favor individuals that maximize the netbenefits of the interaction and minimize the costs. Larvae ofmany species of lycaenid butterflies secrete nutritious foodrewards to attending ants and, in return, receive protectionagainst predators and parasitoids. Because ants typically recruitmore workers to larger resources, by forming groups the larvaemay ensure more reliable access to ants and thereby gain betterprotection. A further consequence of aggregating should be achange of the cost-benefit relationship for individual larvae.The larger the group, the smaller a single larva's influencewill be on total ant density, which could lead to a smallerinvestment in secretion, thus reducing the per capita cost ofcooperation. In this study, die influence of ant attendance,group size, and companion quality on larval investment was investigated.The interaction between the obligately ant-dependent lycaenid,Jalmanus evagoras, and its attendant Iridomyrmax ants was manipulatedand the effect on larval secretion measured. As the level ofant attendance increased, the delivery of food rewards increased,bodi for solitary and for aggregated larvae. When aggregated,larvae provided less food rewards to ants dun when solitary,and secretion rate decreased with increasing group size. Furthermore,larvae had lower secretion rates when paired with a bigger,more attractive larva than when paired with a smaller one. Theconsiderable reduction in secretion rates for larvae in groupssuggests that gaining protection at a lower secretion cost couldbe one factor that promotes aggregation in myrmecophilous lycaenids.     

Specificity of an ant-lycaenid interaction

Summary Many lycaenid butterflies are believed to be mutualists of ants — the butterfly larvae secrete sugars and amino acids as rewards for the ants, and the ants protect the larvae from predation or parasitism. We examined the specificity of the relationship between the lycaenid Plebejus argus and ants in the genus Lasius. Eggs were not attractive to Lasius ants until the emerging larvae had broken through the chorion. First instar larvae were palpated and picked up by Lasius workers and taken to the nest. First instars were mostly ignored by Myrmica sabuleti ants and they were rarely detected by Formica fusca. Older larvae were more attractive to Lasius than to the other ant genera. Pupae were very attractive to Lasius, moderately so to Myrmica, and were ignored by Formica fusca. Teneral adults were palpated by Lasius, but were attacked by Myrmica and Formica workers. We conclude that P. argus is a specialist associate of Lasius ants. Two populations of Plebejus argus were compared: one is naturally associated with Lasius niger, and the other with Lasius alienus. In reciprocal trials, larvae were slightly more attractive to their natural host ant species. Since test larvae were reared on a single host plant species in captivity, this differentiation probably has a genetic basis.     

Ants,plant characteristics and habitat conservation status affect the occurrence of myrmecophilous butterflies on an extrafloral nectaried Malpighiaceae

We investigated the occurrence of myrmecophilous florivorous lycaenid larvae in Banisteriopsis malifolia (Malpighiaceae) according to habitat conservation status (disturbed and preserved savanna), plant phenology, height and the presence of tending ants. The abundance and richness of lycaenids were sixfold and fivefold greater, respectively, in the disturbed area than in the protected savanna. Lycaenids occurred mostly on plants visited by Camponotus blandus, a mutualistic partner of larvae. Habitat type was the main factor influencing lycaenid occurrence, as plants in open areas offered more food resources and tending ants. Banisteriopsis malifolia was considered useful as a host for lycaenids in disturbed sites.