<Emphasis Type="Italic">Virola sebifera</Emphasis> Aubl. (Myristicaceae) leaf chemical composition and implications on leaf-cutter ant foraging choice

Leaf-cutter ant plant material choice is essential for colony maintenance and growth. Plant material is used as a substrate for cultivating symbiotic fungus, and the ants’ preference for particular leaves, tends to be determined by vegetal age-related physicochemical factors. The plant species Virola sebifera Aubl. (Myristicaceae), for example, shows a large number of leaf surface trichomes. Although non-glandular, V. sebifera trichomes may gradually retain an increasing amount of chemical compounds over the lifetime of the leaf. Thus, the present study aims to investigate the role of plant chemical compounds on Atta sexdens rubropilosa preference for V. sebifera leaves of different ages. For this purpose, the chemical composition of trichomes on young and senescent leaves was analyzed, and ants’ preference tested. The chemical compositions differ between V. sebifera young and senescent leaves, with triacontane (C30) predominance in young leaves and tetratriacontane (C34) predominance in senescent leaves. Ants’ preference choice was tested by randomly offering leaves of different ages to A. sexdens rubropilosa workers, following six different treatments: (1) young leaf fragments; (2) young leaf fragments with few trichomes removed; (3) loose trichomes from young leaves; (4) senescent leaf fragments; (5) senescent leaf fragments with few trichomes removed, and (6) loose trichomes from senescent leaves. Ants’ preference was observed for young leaves fragments with a few trichomes removed and also for young leaves loose trichomes. Ants’ preference might be due to specific volatile compounds (GLV) preset in V. sebifera young leaves. Results suggest occurrence of ants’ selectivity resulting from changes on trichomes chemical composition between V. sebifera leaves different age stages.     

Lethal Effect and Behavioral Responses of Leaf-Cutting Ants to Essential Oil of <Emphasis Type="Italic">Pogostemon cablin</Emphasis> (Lamiaceae) and Its Nanoformulation

Leaf-cutting ants belonging to the genus Atta (Formicidae: Myrmicinae) are important pests in agricultural and forest environments. In the present study, we evaluated the formicidal activity of the essential oil of Pogostemon cablin and its nanoformulation on the leaf-cutting ants: Atta opaciceps (Borgmeier, 1939), Atta sexdens (Linnaeus, 1758), and Atta sexdens rubropilosa Forel, 1908. The nanoformulation was developed by magnetic stirring using polyoxyethylene (36%), pure ethanol (36%), essential oil of P. cablin (18%), and water (10%). Bioassays of acute toxicity by fumigation and behavioral bioassays in treated arenas, with and without choice, were performed. The essential oil of P. cablin and its nanoformulation demonstrated efficient insecticidal activity and irritability to ant species. The concentration required to kill 50% of workers varied from 1.06 to 2.10 μL L^?1, with a mean time to death of less than or equal to 42 h. The essential oil of P. cablin and its nanoformulation reduced the displacement and velocity speed of the workers of A. opaciceps and A. sexdens rubropilosa in totally treated arenas. In the bioassays with choices, the three species of ants walked less and at a greater speed on the treated side of arena. This work demonstrates the potential of the essential oil of P. cablin and its nanoformulation to the generation of new formicidal products.     

Ants Do Not Traverse the Silk of Adult Female <Emphasis Type="Italic">Nephila clavipes</Emphasis> (Linnaeus) Webs

Many organisms use chemicals to deter enemies. Some spiders can modify the composition of their silk to deter predators from climbing onto their webs. The Malaysian golden orb-weaver Nephila antipodiana (Walckenaer) produces silk containing an alkaloid (2-pyrrolidinone) that functions as a defense against ant invasion—ants avoid silk containing this chemical. In the present study, we test the generality of ants’ silk avoidance behavior in the field. We introduced three ant species to the orb webs of Nephila clavipes (Linnaeus) in the tropical rainforest of La Selva, Costa Rica. We found that predatory army ants (Eciton burchellii Westwood) as well as non-predatory leaf-cutting ants (Atta cephalotes Linnaeus and Acromyrmex volcanus Wheeler) avoided adult N. clavipes silk, suggesting that an additional species within genus Nephila may possess ant-deterring silk. Our field assay also suggests that silk avoidance behavior is found in multiple ant species.     

Reduced ant defenses in <Emphasis Type="Italic">Macaranga</Emphasis> myrmecophytes (Euphorbiaceae) infested with a winged phasmid <Emphasis Type="Italic">Orthomeria cuprinus</Emphasis>

Macaranga is a tree genus that includes many species of myrmecophytes, which are plants that harbor ant colonies within hollow structures known as domatia. The symbiotic ants (plant–ants) protect their host plants against herbivores; this defense mechanism is called ‘ant defense’. A Bornean phasmid species Orthomeria cuprinus feeds on two myrmecophytic Macaranga species, Macaranga beccariana and Macaranga hypoleuca, which are obligately associated with Crematogaster ant species. The phasmids elude the ant defense using specialized behavior. However, the mechanisms used by the phasmid to overcome ant defenses have been insufficiently elucidated. We hypothesized that O. cuprinus only feeds on individual plants with weakened ant defenses. To test the hypothesis, we compared the ant defense intensity in phasmid-infested and non-infested M. beccariana trees. The number of plant–ants on the plant surface, the ratio of plant–ant biomass to tree biomass, and the aggressiveness of plant–ants towards experimentally introduced herbivores were significantly lower on the phasmid-infested trees than on the non-infested trees. The phasmid nymphs experimentally introduced into non-infested trees, compared with those experimentally introduced into phasmid-infested trees, were more active on the plant surface, avoiding the plant–ants. These results support the hypothesis and suggest that ant defenses on non-infested trees effectively prevent the phasmids from remaining on the plants. Thus, we suggest that O. cuprinus feeds only on the individual M. beccariana trees having decreased ant defenses, although the factors that reduce the intensity of the ant defenses remain unclear.     

Interaction between a threatened endemic plant (<Emphasis Type="Italic">Anchusa crispa</Emphasis>) and the invasive Argentine ant (<Emphasis Type="Italic">Linepithema humile</Emphasis>)

Seed dispersal mutualisms are essential to ensure the survival of diverse plant species and communities worldwide. Here, we investigated whether the invasive Argentine ant can replace native ants by fulfilling their functional role in the seed dispersal of the rare and threatened endemic myrmecochorous plant, Anchusa crispa, in Corsica (France). Our study addressed the potential of Linepithema humile to disperse elaiosome-bearing seeds of A. crispa, examining L. humile’s effects on (1) the composition of communities of ants removing seeds, (2) the number of seed removals, (3) seed preference, (4) the distance of seed dispersion, and (5) seed germination. We caught seven native species at the control site, but only the Argentine ant at invaded sites. L humile removed A. crispa seeds in greater numbers than did native ants, respectively 66 and 23%, probably due to their higher worker density. The invader was similar to native ants with respect to distance of seed transport. Finally, rates of seed germination were not significantly different between seeds previously in contact with either Argentine ants or not. Taken all together, these results suggest that the Argentine ant is unlikely to pose a threat to A. crispa population. These results have important implications for the management of this rare and threatened endemic plant and provide an example of non-negative interactions between invasive and native species.     

Comparative analysis of fungal communities in colonies of two leaf-cutting ant species with different substratum preferences

Fungus gardens of leaf-cutting ants harbor diverse alien fungi in addition to their fungal cultivar. Previous work suggested that alien microorganisms are likely derived from the substrata foraged by ant workers and incorporated into the fungus gardens. To test this hypothesis, we sampled 1014 garden fragments from 16 field colonies of Atta sexdens rubropilosa (a dicot-cutting ant) and Atta capiguara (a grass-cutting ant) in Brazil. From a total of 615 fungal isolates recovered, we observed similar diversity of fungi between colonies of both ant species. However, fungal communities differed in composition of taxa between ant colonies. Trichoderma spirale, Trichosporon chiarellii and Penicillium citrinum were prevalent accounting for 18.5%, 12.2% and 11.7% of the total isolates, respectively. As expected, fungal communities clustered in two major groups supporting the hypothesis that plant substratum has an impact on the composition of the alien fungi found in leaf-cutting ant gardens.     

Leaf-cutting ant fungi produce cell wall degrading pectinase complexes reminiscent of phytopathogenic fungi

Background

Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth.

Results

We tested this hypothesis by using proteomics methods to determine the gene sequences of fecal proteins in Acromyrmex echinatior leaf-cutting ants. Seven (21%) of the 33 identified proteins were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. We show that these enzymes are found in the fecal material only when the ants had access to fungus garden food, and we used quantitative polymerase chain reaction analysis to show that the expression of six of these enzyme genes was substantially upregulated in the fungal gongylidia. These unique structures serve as food for the ants and are produced only by the evolutionarily advanced garden symbionts of higher attine ants, but not by the fungi reared by the basal lineages of this ant clade.

Conclusions

Pectinolytic enzymes produced in the gongylidia of the fungal symbiont are ingested but not digested by Acromyrmex leaf-cutting ants so that they end up in the fecal fluid and become mixed with new garden substrate. Substantial quantities of pectinolytic enzymes are typically found in pathogenic fungi that attack live plant tissue, where they are known to breach the cell walls to allow the fungal mycelium access to the cell contents. As the leaf-cutting ant symbionts are derived from fungal clades that decompose dead plant material, our results suggest that their pectinolytic enzymes represent secondarily evolved adaptations that are convergent to those normally found in phytopathogens.

     

Ants Associated with <Emphasis Type="Italic">Turnera subulata</Emphasis> (Turneraceae): Elaiosome Attraction,Seed Dispersion and Germination

Symbiosis between plants and ants include examples in which the plant provides shelter and/or food for ants that, in turn, act in the defense or in the dispersion of seeds from the host plant. Although traditionally referred as mutualistic, the results of these interactions may vary with the ecological context in which patterns are involved. A range of species have facultative association with Turnera subulata (Turneraceae). Here, using behavioral bioassays, we investigated the effects of the most frequent ant species associated with T. subulata (Brachymyrmex sp.1, Camponotus blandus (Smith), Dorymyrmex sp.1, Crematogaster obscurata Emery, and Solenopsis invicta Buren) in the dispersion of plant host seeds and in the number of seedlings around the associated ant nests. We also evaluated the effects of these ant species in the germination of T. subulata seeds, in the consumption of elaiosome, and in the attractiveness to elaiosome odor. Our results showed that the ant species associated with T. subulata presented variation in the attraction by the odor and in the rate of consumption of the elaiosomes. However, none of the ant species studied contributed significantly to the increase of seed germination and seedling growth. Our results suggest that the consumption of the elaiosome by ant species is not a determinant factor to the success of germination of T. subulata. However, such species could contribute indirectly to seed germination by carrying seeds to sites more fertile to germination. In general, our results help to elucidate the results of ecological interactions involving ants and plants.     

The Behavior of <Emphasis Type="Italic">Acromyrmex crassispinus</Emphasis> (Hymenoptera: Formicidae) on Trail Bifurcations and the Influence of Ant Flow on Error Rates of Nestbound Laden Workers

Ants are ordinarily faced with a succession of bifurcations along their foraging networks. Given that there is no directionality in pheromone trails, each bifurcation is potentially an opportunity for error in the trajectory of laden workers to the nest, which could entail considerable inefficiencies in the transportation of food to the colony. Leaf-cutting ants (Atta and Acromyrmex) commonly show intense traffic and complex foraging trail systems, which make them ideal organisms to study worker behavior in trail bifurcations. The behavior of leaf-cutting ants of the genus Acromyrmex in trail bifurcations is still largely unexplored. Thus, this study aimed to assess the behavior of Acromyrmex crassispinus workers on trail bifurcations and to investigate whether differences in ant flow on foraging trails influence the error rate of nestbound laden workers at trail bifurcation. There was a negative relationship between ant flow and error rate of nestbound laden workers. Most workers walked in the central part of the foraging trails but occupied a broader area of the foraging trail when the ant flow was high. The results of this study provide valuable insight into the organization of traffic flow in A. crassispinus and its impacts on the foraging strategy of the species.     

Mortal combat between ants and caterpillars: an ominous threat to the endangered Schaus swallowtail butterfly (<Emphasis Type="Italic">Heraclides aristodemus ponceanus</Emphasis>) in the Florida Keys,USA

The federally endangered Schaus swallowtail butterfly (Heraclides aristodemus ponceanus) has reached critically low numbers. Exotic ants are a potential threat to H. a. ponceanus and other rare butterflies as they can attack immature stages. Ant surveys conducted in subtropical dry forests in Biscayne National Park documented ant species diversity and relative abundance. A caterpillar predator exclusion experiment using physical barriers in different combinations evaluated caterpillar survivorship of both early and late instar caterpillars exposed to different threats. Ant-caterpillar interactions were also documented by placing caterpillars on plants and observing physical interactions between caterpillars and ants. A total of 1418 ants comprising 25 ant species was captured and identified. In canopies of H. a. ponceanus host plants, 243 ants comprising 12 species were found. The four most common ants collected in the host plant canopies were Pseudomyrmex gracilis, Camponotus planatus, Cremastogaster ashmeadi, and Camponotus floridanus. The predator exclusion experiment revealed survivorship was significantly lower for early and late instar caterpillars without any physical barrier, as well as for early instars not protected by a mesh cage. Pseudomyrmex gracilis and C. floridanus were more aggressive towards caterpillars in comparison to other ant species; these two species ranked first and second in the “ant danger index” ranking predatory abilities of the four most common ant species. Pseudomyrmex gracilis is a common arboreal exotic ant in Biscayne National Park and presents a major threat to caterpillars during their earliest life stages.     

Ants (Hymenoptera: Formicidae) as potential egg predators of lepidopteran pests of sweet potato, <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (Solanales: Convolvulaceae), in Japan

The sweet potato Ipomoea batatas (L.) Lam., which has extrafloral nectaries, was visited by nine indigenous ant species in south Kyushu, Japan. These ant species were evaluated for their ability to remove the eggs of two lepidopteran species—Aedia leucomelas (Linnaeus) and Spodoptera litura (Fabricius)—through ant-exclusion experiments. The results showed that Tetramorium bicarinatum (Nylander), one of the dominant ant species, is an efficient predator of lepidopteran eggs. This ant species removed A. leucomelas eggs from ant-visited plants in six trials conducted from mid-July to mid-October, with high removal rates (90–100%) occurring from mid-September onward. On ant-excluded plants, only 0–18% of exposed eggs were removed by ants that accidentally invaded the plants. Tetramorium bicarinatum also removed egg masses and the hatchlings of S. litura. The mean percent area of egg-laden leaves eaten by S. litura larvae was lower on ant-visited plants (0.9%) than on ant-excluded plants (57.3%). In contrast, Formica japonica Motschulsky, another dominant ant species, was not observed removing the eggs or larvae of the lepidopteran species. These results show that certain indigenous ant species are efficient as biocontrol agents. Thus, individual ant species need to be investigated for their ability to control insect pests in various crops.     

Ant-Repelling Pollinators of the Myrmecophytic <Emphasis Type="Italic">Macaranga winkleri</Emphasis> (Euphorbiaceae)

Many plants have mutualistic relationships with ants, whereby plants provide food and/or nesting sites for the symbiotic ants, and in turn the ants protect the host plants by excluding herbivores. While the ants are useful as guards, they may negatively affect host reproduction by excluding pollinators. Here we studied this potential conflict in the myrmecophytic Macaranga winkleri pollinated by the thrips Dolichothrips fialae. Behavioural responses of ant guards to pollinator thrips and their chemicals, and related chemical analyses, provide evidence that thrips deter ant-guards by secreting droplets containing ant-repelling n-decanoic acid from their anuses. This is the first report of insect pollinators repelling their host’s symbiotic guard ants to perform pollination. This is a novel strategy by which a plant host avoids interference with pollination by ant-guards in an ant–plant mutualism. The acquisition of a pollination system that is resistant to ant attacks may have facilitated the evolution of myrmecophytes in the genus Macaranga.     

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.     

Mass Occurrence and Dominant Behavior of the European Ant Species <Emphasis Type="Italic">Formica fuscocinerea</Emphasis> (Forel)

Recently, masses of the ant Formica (Serviformica) fuscocinerea (Forel) have been occurring at numerous sites in Southern Germany. Although F. fuscocinerea is native to Southern Germany, these mass occurrences resemble ant invasions in density and dominance. This study aimed to investigate the underlying mechanisms that promote sudden mass occurrence of a previously inconspicuous ant species within its native range. To estimate the competitive dominance of F. fuscocinerea, species occurrence and abundance considering biotic and abiotic parameters were studied in a natural habitat where F. fuscocinerea co-occurred with two other common ant species, Myrmica ruginodis (Nylander) and Lasius niger (Linnaeus). To understand the species’ distribution in the field, laboratory experiments on interspecific competition were conducted. Finally, the colony structure of F. fuscocinerea was investigated with intraspecific aggression tests. Formica fuscocinerea dominated an area that, as indicated by strongly frequented foraging trails on the trees, provided important food sources, e.g. trophobionts, to the ants. Other ant species coexisted only at the periphery of the F. fuscocinerea range. Laboratory experiments revealed F. fuscocinerea as highly dominant species. Additionally, F. fuscocinerea showed a complete lack of intraspecific aggression between ants originating from distances up to 58 km, indicating weak or nonexistent behavioral boundaries among ants of physically separated nests. Since extraordinarily high worker densities, strong interspecific dominance and a lack of colony boundaries within supercolonies are considered to be important traits of several invasive ant species we conclude that the same traits also promote the dominance of F. fuscocinerea.     

Host ant use of the Alcon blue butterfly at the northern range margin

In myrmecophilous insects, interactions with ants are often a key factor determining persistence of their populations. Regional variation in host ant use is therefore an essential aspect to consider to provide adequate conservation practices for such species. In this study, we examined this important facet of species’ ecology in an endangered myrmecophilous butterfly Phengaris (=Maculinea) alcon (Lepidoptera, Lycaenidae). The investigations conducted in peripheral populations in Estonia allowed us to expand the knowledge of its host ant use to the northern distribution limit of the species. Our data indicate that in its northernmost populations, the xerophilous ecotype of Phengaris alcon is primarily parasitizing a single host ant species, Myrmica schencki. The data collected are in line with the emerging evidence suggesting that peripheral and core populations of P. alcon use different host ants, and peripheral populations tend to display higher host ant specificity. We also show that, at its northern range margin, P. alcon might be more limited by the availability of its sole larval food plant in the region, Gentiana cruciata, than the densities of its host ant. Finally, we found a strong negative correlation between Myrmica spp. and Lasius spp. colony densities, suggesting that interspecific competition between ants could have a substantial influence on host ant availability of Phengaris butterflies, and thus should be taken into account in conservation plans of these species.     

Phenotypic and phylogenetic characterization of actinomycetes isolated from <Emphasis Type="Italic">Lasius niger</Emphasis> and <Emphasis Type="Italic">Formica cunicularia</Emphasis> ants

Multiple actinomycete strains were isolated from two ant species, Lasius niger and Formica cunicularia, and their phenotypic properties and phylogenetic position were studied. Partial sequencing of 16S rRNA assigned the greater part of them to the genus Streptomyces, but only one belonged to Nocardia. However, some isolates had significant color and morphological differences from their closest phylogenetic relatives. The abundance and biodiversity of actinomycete communities isolated from L. niger ants greatly exceeded those found for F. cunicularia. All of the actinomycetes associated with F. cunicularia ants demonstrated cellulolytic activity, but only one had such ability among the strains associated with black ants.     

Ant-partners play a minor role on occurrence of the myrmecophilous butterfly <Emphasis Type="Italic">Leptotes cassius</Emphasis> in its host plant

Ant-related oviposition in facultatively myrmecophilous lycaenid butterflies is common, but not universal. In fact, our knowledge of ant-related oviposition in lycaenids is based on some common species (e.g., Rekoa marius, Allosmaitia strophius, Parrhasius polibetes), which limits generalizations about these systems. In this study, we experimentally investigated whether the oviposition pattern of the florivorous lycaenid Leptotes cassius was influenced by the presence of Camponotus ants and whether larvae were attended, rather than attacked, by ants. This might be evidence of myrmecophily. Both L. cassius and Camponotus ants occur on Bionia coriacea, an extrafloral nectaried legume shrub that grows in the Brazilian cerrado. Plants were randomly assigned to ant-present and ant-excluded treatments and were observed twice throughout the short reproductive season. Larvae of L. cassius were tended by ants, whose attendance was characterized by active antennation on the last body segments of the caterpillars. Therefore, Camponotus can be considered a partner of L. cassius. Lycaenid abundance was on average 1.9- and 1.21-fold higher in plants with ants in each sampling period, respectively, indicating a tendency of L. cassius to occur in plants with ants. Nonetheless, results were not statistically significant, suggesting that in this case ants are not a major cue for lycaenid oviposition. In many ant–lycaenid mutualisms, butterfly immatures benefit from reduced parasitism rates. However, no L. cassius immature, regardless of ant presence or absence, was parasitized. Furthermore, larvae may occur inside flower buds that may provide protection from natural enemies; thus, ants may not be required for immature protection.     

Complex flowers and rare pollinators: Does ant pollination in <Emphasis Type="Italic">Ditassa</Emphasis> show a stable system in Asclepiadoideae (Apocynaceae)?

If on one hand, ant pollination is rare, on the other Asclepiadoideae flowers are the most complex among Eudicots. Both themes are exciting in pollination biology. Although there are records of ants with the ability to remove the pollinarium on this subfamily, the role of these insects as pollinators is not yet known. Therefore, we investigated the interaction between flowers and ants, as well as the associated features in two species of Asclepiadoideae, Ditassa capillaris and D. hastata. The studied species were both visited by Cephalotes inaequalis and Dorymyrmex thoracicus. We analyzed the common traits of ant pollination of the plant species, recorded the number of visit by ants, and carried out experiments of selective pollination, developing an effectivity index. Both Ditassa species have common traits of ant pollination, such as small bright flowers with exposed floral resource. The pollinarium is clip-shaped and resistant to the metapleural gland, which ensures effective pollination. Dorymyrmex thoracicus is a very frequent but not very efficient pollinator, whereas Cephalotes inaequalis is more efficient though at a lower frequency. Therefore, the total contributions of both ants to the pollination of both Ditassa species are similar. The features of both Ditassa species which favor ant pollination, include some synapomorphies within Asclepiadoideae. Furthermore, the efficiency of the ants as pollinators to the reproductive success of the plants was similar despite the different behaviors observed in both ant species. Hence, we suggest that the role of ants in pollination within this subfamily has been neglected.