Neotropical ant gardens II. Bioassays of seed compounds

A number of volatile compounds occur on the seeds of taxonomically unrelated ant-garden epiphytes in western Amazonia. In field trials in southeastern Peru, we assayed the responses of ant-garden ants (Camponotus femoratus) to these and structurally similar compounds applied to artificial seeds made from zeolite molecular sieves. Benzothiazole,2, present on seeds of eight ant-garden epiphytes, repelled ants over the range of concentrations tested, as did 1-(2-hydroxy-6-methylphenyl)ethanone,3, occurring on seeds of six ant-garden epiphytes. 2-Hydroxy-6-methylbenzoic acid, methyl ester (methyl-6-methylsalicylate, 6-MMS),1, found on seeds of at least nine ant-garden epiphytes, was mildly repellent at high concentration, but stimulated excitement, seed handling, and (rarely) seed carrying at lower concentrations. Vanillin,5, a seed compound of four ant-garden epiphytes, and limonene,6, a monoterpene from seeds of three ant-garden epiphytes, both stimulated excitement, alarm, seed handling, and (rarely) seed carrying. Identified from seeds of seven ant-garden epiphytes, 1-(2,4-dihydroxyphenyl)ethanone,4, elicited little or no response. Among 70 compounds tested (mainly aromatic compounds), those found on seeds of ant-garden epiphytes or having structural features in common with such compounds were the most attractive to the ants. Although not present on epiphyte seeds, 2-hydroxy-3-methoxybenzenemethanol,10, consistently stimulated seed transport to the nest in one year, but did so only rarely in subsequent years. Some of the volatile compounds on seeds of ant-garden epiphytes probably play a role in ant attraction to epiphyte seeds, but evidence remains ambiguous. Finally,Ca. femoratus responded to one test compound [1-(2-hydroxy-4-methoxyphenyl)ethanone,60] (absent from epiphyte seeds) by descending from the vegetation to the ground.     

Antifungal Diketopiperazines from Symbiotic Fungus of Fungus-Growing Ant Cyphomyrmex minutus

The attine fungus Tyridiomyces formicarum, the symbiont of the fungus-growing ant Cyphomyrmex minutus, produces several antifungal diketopiperazines. This represents the first identification of antifungal compounds from an attine symbiont and contradicts previous suggestions that attine fungi do not produce metabolites with antifungal activity. T. formicarum probably produces antifungal compounds in defense (1) against other fungi that invade the gardens and escape the weeding activity of the ants, or (2) against ant-pathogenic fungi that could harm the host ants. Fungi cultivated by fungus-growing ants may represent a rich source of additional bioactive metabolites.     

Queen Sex Pheromone of the Slave-making Ant, Polyergus breviceps

Workers of the slave-making ant, Polyergus breviceps, raid nests of Formica ants and return with Formica pupae that mature into worker ants in the slave-makers’ colony. These Formica workers then tend the Polyergus brood, workers, and reproductives. During raids in the mating season, winged virgin Polyergus queens accompany the workers in the raiding columns. During the raid, the virgin queens release a pheromone that attracts males that quickly mate with the queens. We report the identification, synthesis, and bioassay of the sex attractant pheromone of the queens as an approximately 1:6 ratio of (R)-3-ethyl-4-methylpentan-1-ol and methyl 6-methylsalicylate. The ants produce exclusively the (R)-enantiomer of the alcohol, and the (S)-enantiomer has no biological activity, neither inhibiting nor increasing attraction to blends of methyl 6-methylsalicylate with the (R)-enantiomer.     

The Role of Extrafloral Nectar Amino Acids for the Preferences of Facultative and Obligate Ant Mutualists

Plants in some 300 genera produce extrafloral nectar (EFN) to attract ants as a means of indirect defence. Among Mesoamerican Acacia species, obligate myrmecophytes produce EFN constitutively to nourish symbiotic ant mutualists, while non-myrmecophytes induce EFN secretion in response to herbivore damage to attract non-symbiotic ants. Since symbiotic Acacia ants entirely depend on the host-derived food rewards while non-symbiotic ants need to be attracted to EFN, this system allows comparative analyses of the function of EFN components in ant nutrition and attraction. We investigated sugar and amino acid (AA) composition in EFN of two myrmecophytes (Acacia cornigera and Acacia hindsii) and two related non-myrmecophyte species (Acacia farnesiana and Prosopis juliflora). AA composition allowed a grouping of myrmecophytes vs. non-myrmecophytes. Behavioural assays with obligate Acacia inhabitants (Pseudomyrmex ferrugineus) and non-symbiotic ants showed that AA composition affected ant preferences at high but not at low AA/sugar ratios. Most interestingly, behavioural responses differed between the two types of ants tested: Symbiotic ants showed a clear preference for higher AA concentrations and preferred nectar mimics with those four AAs that most significantly characterised the specific nectar of their Acacia host plant. In contrast, non-symbiotic ants distinguished among nectars containing different sugars and between solutions with and without AAs but neither among nectars with different AA/sugar ratios nor among mimics containing different numbers of AAs. Our results confirm that both AAs and sugars contribute to the taste and attractiveness of nectars and demonstrate that the responses of ants to specific nectar components depend on their life style. AAs are a chemical EFN component that likely can shape the structure of ant–plant mutualisms.     

Chemical Ecology of the Defense of Two Nymphalid Butterfly Larvae Against Ants

We analyzed the behavioral responses of the ants Camponotus rufipes and Solenopsis geminata towards all instars of Dione junio and Abananote hylonome. We also analyzed ant behavior towards hexane extracts of larvae and extracts of the spines and neck glands of the fifth instars of both species and identified the chemical compounds present. Larvae of both species were repellent to ants from the first instar onward. Later instars survived ant attacks better than earlier instars. The spines and neck glands of the larvae influenced the behavior of C. rufipes. The chemical compounds contained in the hexane extracts of whole first and fifth instars and in the spines and neck glands of fifth instars were principally carboxylic acids and terpenes. Further bioassays confirmed the repellent effect of some of these acids toward ants.     

Food experience on the predatory behavior of the ant Myrmica rubra towards a specialist moth,Acrolepiopsis assectella

Entomophagous insects are often repelled by the secondary compounds of the plants eaten by their prey. These compounds, therefore, take on a defensive role for the phytophagous species that sequester them. Given that numerous entomophagous species are capable of learning, the effects on the foraging behavior of a repeated experience were investigated in the predatory ant Myrmica rubra. The sulfur amino acids methyl-cysteine sulfoxide (MCSO) and propyl-cysteine sulfoxide (PCSO) produced by Allium plants were identified in caterpillars of the leek moth Acrolepiopsis assectella. Three behavioral studies were carried out, with or without prior familiarization with caterpillars reared either on leek or on an artificial diet containing no Allium compounds. In choice tests with the two types of caterpillars, unfamiliarized ants displayed a preference for caterpillars reared on the artificial diet, but this preference disappeared or was reversed in both young and old ants after familiarization.     

Behavioral and electrophysiological studies with live larvae and larval rinses of the red imported fire ant,Solenopsis invicta Buren (Hymenoptera: Formicidae)

Behavioral and electrophysiological studies with live intact larvae and larval rinses of the red imported fire ant,Solenopsis invicta Buren, give undeniable evidence of a volatile material associated with the larvae of the ant that is capable of eliciting a response from brood-tending workers. In a Y-tube bioassay, worker ants were attracted equally to an airstream blown over sibling larvae or heterocolonial larvae. Workers were also attracted to a rinse of the larvae in a spot bioassay, aggregated about a piece of surrogate brood in another bioassay, and retrieved surrogate brood treated with the rinse material. A dose-response curve constructed from electroantennograms of workers revealed a receptor response of 1–100 brood equivalents.     

Defensive role ofAllium sulfur compounds for leek mothAcrolepiopsis assectella Z. (Lepidoptera) against generalist predators

It has been shown previously that sulfur volatiles produced byAllium plants affect the behavior of their specialist phytophages and of their specialist entomophages. The action of these compounds in protecting the leek mothAcrolepiopsis assectella against generalist entomophages was studied in comparison to the proposed original defensive role of these compounds against generalist herbivorous insects. Two ants species,Formica selysi andF. fusca, were used as generalist predators. Six behavioral criteria of the predatory behavior of the ants were studied in presence of the last-instar caterpillars (C₅). C₅ reared on artificial diets with or without leek components were tested, as well as C₅ soaked in frass of leek-reared caterpillars or disulfide solutions. In addition, the response of the ants to pure chemicals found in leek was studied using honey solutions with or without sulfur compounds. The sulfur allelochemicals ofAllium plants have a negative action on predatory ants. Interestingly, the nonvolatile precursors of sulfur volatiles ofAllium plants seem to have a protective role for their phytophagous insects against generalist entomophages.     

3-Hexyl-5-Methylindolizidine Isomers from Thief Ants, Solenopsis (Diplorhoptrum) Species

The venom alkaloids from the workers of nine collections of Solenopsis (Diplorhoptrum) from California contain either (5E,9E)-3-hexyl-5-methylindolizidine (1c) or (5Z,9E)-3-hexyl-5-methylindolizidine (1d) along with cis-2-methyl-6-nonylpiperidine. The structures of these compounds were determined from their mass spectra and by comparison of their GC-FTIR spectra with those of a synthetic mixture. In view of the facts that a third diastereomer of 3-hexyl-5-methylindolizidine had been reported in previous collections of Solenopsis (Diplorhoptrum) queens from Puerto Rico, and that indolizidines along with other ant venom alkaloids are sequestered by amphibians, the determination of species in this difficult group of ants is significant. In particular, the chemotaxonomic value of the stereochemistry of these venom alkaloids is discussed.     

Larval Beetles Form a Defense from Recycled Host-Plant Chemicals Discharged as Fecal Wastes

Larvae of the leaf-feeding beetles Neolema sexpunctata and Lema trilinea carry feces on their backs that form shields. We used the generalist predatory ant, Formica subsericea, in a bioassay to determine whether shields were a physical barrier or functioned as a chemical defense. Fecal shields protected both species against ant attack. Larvae of both species reared on lettuce produced fecal shields that failed to deter ants. Commelina communis, N. sexpunctata's host, lacks noxious secondary compounds but is rich in phytol and fatty acids, metabolites of which become incorporated into the fecal defense. In contrast, the host plant of L. trilinea, Solanum dulcamara, contains steroidal glycoalkaloids and saponins, whose partially deglycosylated metabolites, together with fatty acids, appear in Lema feces. Both beetle species make modifications to host-derived precursors before incorporating the metabolites into shields. Synthetic chemicals identified as shield metabolites were deterrent when applied to baits. This study provides experimental evidence that herbivorous beetles form a chemical defense by the elimination of both primary and secondary host-derived compounds. The use of host-derived compounds in waste-based defenses may be a more widely employed strategy than was hitherto recognized, especially in instances where host plants lack elaborate secondary compounds.     

Reaction of Mutualistic and Granivorous Ants to Ulex Elaiosome Chemicals

It has been proposed that chemicals on plant elaiosomes aid seed detection by seed-dispersing ants. We hypothesized that the chemical interaction between ants and elaiosomes is more intimate than a generic attraction, and that elaiosome chemicals will attract mutualistic but not granivorous ant species. We investigated this by using two gorse species, Ulex minor and U. europaeus, and two associated ant species from European heathlands, the mutualist Myrmica ruginodis and the granivore Tetramorium caespitum. Behavioral studies were conducted with laboratory nests and foraging arenas. Both ants will take Ulex seeds, but while M. ruginodis showed increased antennation toward ether extracts of elaiosome surface chemicals compared with controls, T. caespitum showed no response. Elaiosome extracts were separated into seven lipid fractions. M. ruginodis showed increased antennation only toward the diglyceride fractions of both Ulex species, whereas T. caespitum showed no consistent reaction. This indicates that M. ruginodis can detect the elaiosome by responding to its surface chemicals, but T. caespitum is unresponsive to these chemicals. Responses to surface chemicals could increase the rate of seed detection in the field, and so these results suggest that Ulex elaiosomes produce chemicals that facilitate attraction of mutualistic rather than granivorous ant species. This could reduce seed predation and increase Ulex fitness.     

Diaspore Trait Preferences of Dispersing Ants

Elaiosomes of myrmecochorous plant seeds are known to enhance the attraction of diaspore-dispersing ants by serving as a nutritional reward. However, it remained unclear which (nutritional) compounds affect diaspore preferences of ants. We hypothesized that apart from elaiosome/seed-size ratio, volume, and physical surface of diaspores, the quantity and the composition of fatty acids, amino acids, and sugars strongly influence the diaspore preferences of different species. Chemical (nutritional) profiles as well as structural properties of seeds with and without elaiosomes were analyzed and correlated with observed seed choice behavior of ants. Cafeteria experiments in the field confirmed the enhanced attractiveness of elaiosome-bearing seeds for all three ant species tested (Lasius fuliginosus, Myrmica ruginodis, and Temnothorax nylanderi), although seeds lacking elaiosomes also were transported. In multiple-choice cafeteria experiments with simultaneously offered diaspores of 16 plant species with and without elaiosome and with highly varying structural and chemical properties, all three ant species showed distinct preferences for certain diaspore species. Correlation analyses confirmed that the presence of an elaiosome represents the crucial factor that favors ant diaspore dispersal. In addition, the composition and the content of free amino acids, and to varying degrees fatty acids, were found to significantly affect preferences of each ant species, whereas the effect of single fatty acids acting as chemical triggers for diaspore transport by ants, as supposed by several studies, was not confirmed. In conclusion, although at least some diaspore species lacking elaiosomes attract ants for diaspore removal services by presenting nutritional seed coats, the production of elaiosomes seems to provide a worthwhile investment. Elaiosomes ensure rapid diaspore detection and removal due to chemical cue compounds and by offering a highly nutritional food supply, probably fitting the nutritional demands of ants.     

Responses by amphisbaenianBlanus cinereus to chemicals from prey or potentially harmful ant species

We tested the ability of amphisbaenians (Blanus cinereus) to discriminate between odors of ant species selected as prey (Pheidole pallidula) and odors of potentially harmful ant species (Messor barbarus) that are avoided. Tongue-flick rate to swabs impregnated with ant odors, cologne, or deionized water differed among treatments, showing that amphisbaenians were able to discriminate ant species odors. Amphisbaenians showed an aggressive response and bit applicators bearing the odor of harmful ants, while the odor of prey ants did not elicit bites to swabs. The possible evolutionary advantage of identifying and avoiding harmful ants is discussed in relation to the fossoriality of amphisbaenians.     

<Emphasis Type="Italic">E</Emphasis>-2-Ethylhexenal, <Emphasis Type="Italic">E</Emphasis>-2-Ethyl-2-Hexenol,Mellein, and 4-Hydroxymellein in <Emphasis Type="Italic">Camponotus</Emphasis> Species from Brunei

E-2-ethyl-2-hexen-1-ol (1), mellein (4), and 4-hydroxymellein (5) were identified as the major volatile compounds in the head and/or thorax of Camponotus quadrisectus. Neither 1 nor 5 have been previously detected in insects. Also identified were small amounts of m-cresol (2) and 6-methyl salicylic acid (3). E-2-ethylhexenal (6) and small amounts of 3 were identified in heads of Camponotus irritibilis from Kuala Belalong, Brunei. Compounds 2–4 occur in other Bornean camponotines with hypertrophied mandibular glands, and 4 is widespread in the tribe. The possibility of semiochemical parsimony (multiple functions) for these mandibular gland compounds is reviewed in the context of existing data on mandibular gland products of other camponotines, reported biological activities of the compounds, and secondary loss of metapleural glands in this ant group.     

Species-specific Leaf Volatile Compounds of Obligate <Emphasis Type="Italic">Macaranga</Emphasis> Myrmecophytes and Host-specific Aggressiveness of Symbiotic <Emphasis Type="Italic">Crematogaster</Emphasis> Ants

Macaranga myrmecophytes harbor species-specific Crematogaster ants that defend host trees from herbivores. We examined ant aggressive behaviors when artificially damaged leaf pieces from another tree were offered to four sympatric species of obligate Macaranga myrmecophytes. The ants showed aggressive behavior in response to leaf pieces regardless of the leaf species; however, aggressiveness was higher when conspecific leaf pieces were offered than when nonhost species were offered. Thus, ants can recognize leaf damage and distinguish among damaged leaf species. Chemical analyses of volatile compounds emitted from damaged leaves that may induce ant defense showed that the composition of the minor compounds differed among the four Macaranga species, although there were many compounds in common.     

Trade-Off Between Chemical and Biotic Antiherbivore Defense in the South East Asian Plant Genus Macaranga

The plant genus Macaranga is known for its manifold mutualistic associations with ants. The plants provide food for the ants and in turn get protection from herbivores. Depending on the strength of the plant–ant interaction, the plant's investment in ants and the biotic defense derived from them is more or less effective. We conducted a comparative study on tannin content in 12 Macaranga species that were selected based on their associations with ants (three nonmyrmecophytes and nine myrmecophytes, three of which start their ontogeny as nonmyrmecophytes). Different developmental stages were investigated in three Macaranga species. Extracts of every individual plant analyzed for tannins were also tested for their effects on larval growth employing larvae of the common cutworm (Spodoptera littoralis). The studied Macaranga species differed significantly in their tannin contents as well as in the effects of their leaf extracts on the growth of S. littoralis larvae. A correlation analysis shows a connection between tannin contents and larval growth. High tannin contents and, thus more effective chemical defense, were observed in nonmyrmecophytic Macaranga species associated only facultatively with ants as compared to obligate myrmecophytes. Our study supports the hypothesis of a trade-off between chemical and biotic defense in the genus Macaranga.     

Mellein, a Trail Pheromone Component of the Ant Lasius fuliginosus

3,4-Dihydro-8-hydroxy-3-methylisocoumarin (mellein) and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one are among the volatile constituents identified from the hindgut of the formicine ant Lasius (Dendrolasius) fuliginosus Mellein induces trail-following behavior in worker ants of this species and evokes electrophysiological responses from their antennae. The trail-following activity released by (R)-(–)-mellein is significantly higher than that elicited by its (S)-(+) antipode, or the racemic mixture. The above-mentioned pyranone is found also in the honeydew of aphids, the customary diet of this ant species. The pyranone also evokes some trail-following behavior, but its activity is far less pronounced than that of mellein. Apparently, extra pheromone components are present in the hindgut, since the activity of these two constituents, either individually or as a mixture, does not completely account for the total activity released by a hindgut extract containing similar amounts of these two compounds.     

The Effects of Ants on the Entomophagous Butterfly Caterpillar Feniseca tarquinius, and the Putative Role of Chemical Camouflage in the Feniseca–Ant Interaction

Butterfly caterpillars in the lycaenid subfamily Miletinae are predators of ant-tended Homoptera, yet they lack specialized secretory and call-production organs crucial to ant association in other lycaenids. Here, we address the question of how miletine caterpillars have invaded the ant–Homoptera symbiosis through a study of the only New World miletine, Feniseca tarquinius, a predator of the wooly aphid Prociphilus tesselatus. Previous interpretations have suggested that F. tarquinius and other miletine caterpillars avoid ant aggression by concealing themselves under silken webs. In contrast, our field data indicate that F. tarquinius caterpillars are less likely to be concealed in the presence of the ants Camponotus pennsylvanicus and Formica obscuriventris than in the absence of ants, although caterpillar and ant behaviors vary between years. Chemical analysis and behavioral assays suggest that chemical camouflage, not physical concealment, is responsible for the ants’ failure to detect and remove F. tarquinius caterpillars from aphid colonies. Analyses by gas chromatography indicate that the cuticular lipid composition of caterpillars are similar to that of their aphid prey, although it varies with prey species. Behavioral assays confirm that solvent extracts of F. tarquinius caterpillars and P. tesselatus aphids evoke similar behavioral responses in C. pennsylvanicus ants. Chemical camouflage is well known in social parasites of ants, but the present study represents one of a few documented cases where chemical deceit is important to interactions with ants outside the nest.     

Synergistic Effects of Three Piper Amides on Generalist and Specialist Herbivores

The tropical rainforest shrub Piper cenocladum, which is normally defended against herbivores by a mutualistic ant, contains three amides that have various defensive functions. While the ants are effective primarily against specialist herbivores, we hypothesized that these secondary compounds would be effective against a wider range of insects, thus providing a broad array of defenses against herbivores. We also tested whether a mixture of amides would be more effective against herbivores than individual amides. Diets spiked with amides were offered to five herbivores: a naïve generalist caterpillar (Spodoptera frugiperda), two caterpillar species that are monophagous on P. cenocladum (Eois spp.), leaf-cutting ants (Atta cephalotes), and an omnivorous ant (Paraponera clavata). Amides had negative effects on all insects, whether they were naïve, experienced, generalized, or specialized feeders. For Spodoptera, amide mixtures caused decreased pupal weights and survivorship and increased development times. Eois pupal weights, larval mass gain, and development times were affected by additions of individual amides, but increased parasitism and lower survivorship were caused only by the amide mixture. Amide mixtures also deterred feeding by the two ant species, and crude plant extracts were strongly deterrent to P. clavata. The mixture of all three amides had the most dramatic deterrent and toxic effects across experiments, with the effects usually surpassing expected additive responses, indicating that these compounds can act synergistically against a wide array of herbivores.     

Phytotoxic Iridoid Glucosides from the Roots of Verbascum thapsus

The iridoid glucosides lateroside 1, harpagoside 2, ajugol 3, and aucubin 4 were isolated from an ethanolic extract of the roots of the weed Verbascum thapsus that exhibits antigermination activity on seeds of barley (Hordeum vulgare). Bioassays indicated that at 3 mM concentration, compounds 1, 2, and 4 showed moderate inhibition of seed germination. These compounds also reduced root length when they were assayed on pregerminated seeds at 1 mM to 0.001 mM concentration range. Of all compounds tested, aucubin 4 was the most active against root elongation. Compound 3 showed no activity in the bioassays.