Identification of trail pheromone compounds from the labial glands of the stingless bee Geotrigona mombuca

Foragers of several species of stingless bees deposit pheromone spots in the vegetation to guide recruited nestmates to a rich food source. Recent studies have shown that Trigona and Scaptotrigona workers secrete these pheromones from their labial glands. An earlier report stated that species within the genus Geotrigona use citral from their mandibular glands for scent marking. Since convincing experimental proof for this conjecture is lacking, we studied the glandular origin of the trail pheromone of Geotrigona mombuca. In field bioassays, newly recruited bees were diverted by artificial scent trails that branched off from the natural scent trail deposited by their nestmates only when they were baited with extracts from the foragers’ labial glands. Compounds extracted from the mandibular glands, however, did not release trail following behavior. This demonstrates that the trail pheromone of G. mombuca is produced in the labial glands, as in Trigona and Scaptotrigona. Furthermore, in chemical analyses citral was identified exclusively in the foragers’ mandibular glands, which disproves its supposed role as a trail pheromone. The labial glands contained a series of terpene- and wax type esters, with farnesyl butanoate as major constituent. We, therefore, postulate that the trail pheromone of G. mombuca is composed of a blend of esters.     

Orientation and recruitment behavior in the ponerine ant Pachycondyla tesserinoda (Emery): laying of individual-specific trails during tandem running

Summary Tandem leaders of Pachycondyla tesserinoda mark their way during nest moving. While scouting and foraging for food in an unknown area, chemical orientation is used as well. The origin of the trail substance could not be identified. Secretions of gastral exocrine glands did not induce trail following behavior. Nor do these secretions elicit tandem-following reactions; rather the latter is released by the general body surface odor. The trail substance and the substance used for marking the nest entrance are colony-specific. Moreover, individual tandem leaders recognize and show a preference for their own trails. This extraordinary effect is independent of the age of the trail. P. tesserinoda workers search individually for food and new nest sites. Targets which are important for the colony are directly shown to nestmates by tandem running. Due to this type of foraging and scouting individual-specific trails may be advantageous for this ant.     

A trail pheromone component of the African stink ant, Pachycondyla (Paltothyreus) tarsata Fabricius (Hymenoptera: Formicidae: Ponerinae)

Summary. The African stink ants (Pachycondyla tarsata) lay recruitment trails with secretions from sternal glands. The glandular secretions consist of 10 compounds, 9 of which have been chemically identified. One of the substances, 9-heptadecanone, elicits trail following behavior in P. tarsata workers that have before been stimulated by a sucessful scout ant. Received 7 August 1998; accepted 24 November 1998.     

Structure and function of Dufour gland pheromones from the crazy ant <Emphasis Type="Italic">Paratrechina longicornis</Emphasis>

Summary. Dufour glands of Paratrechina longicornis were analyzed chemically using GC-MS techniques. The glands contained twelve alkanes between C₉ and C₂₀ chain length with undecane and tridecane as the main components, six alkenes (1- and 4-undecene, 1-, 4-, and 6- tridecene, and heptadecadiene), two alkyl formates (C₁₁ and C₁₃), and saturated and polyunsaturated acids. Many of the alkanes and alkenes were behaviorally active causing a short lasting attraction of ants with different intensity. Detection of the major gland compound undecane in recruitment trails by in vivo SPME sampling provided evidence for its use as a recruitment pheromone. Both poison gland (formic acid) and Dufour gland (undecane) contents were detected on SPME fibers which had been attacked by the ants.     

Complex chemical communication in the crazy ant <Emphasis Type="Italic">Paratrechina longicornis</Emphasis> Latreille (Hymenoptera: Formicidae)

Summary. The formicine ant Paratrechina longicornis is known for its extremely opportunistic foraging behaviour. Only a single trail pheromone source, the rectum, was previously described from this ant. Our detailed examination of this ant’s chemical communication system revealed the presence of at least four sources of pheromones. Rectum, poison sac, and Dufour gland contain orientation components with decreasing effectiveness and persistence (in the sequence mentioned) as well as attractants with increasing effectiveness. Furthermore, the mandibular gland contains repellents, and a releaser of defensive behaviour. This set of various signals of different strength and persistence allows an extraordinary degree of flexibility and efficiency in the collective behaviour of P. longicornis, especially food exploitation, and thus may contribute to this insect’s overall ecological success.     

Trail discrimination signal of Lasius japonicus (Hymenoptera: Formicidae)

Summary. Trail-following behavior of Lasius japonicus was colony-specific in the field, while trail pheromone activity was not. We found that the footprint substance caused colony-specific trail-following behavior only when working in conjunction with the trail pheromone. The footprint substance alone did not lead the workers to follow trails. The substance consisted mainly of hydrocarbons with composition almost identical to that of cuticular hydrocarbons, except for the absence of n-alkanes. Nestmate workers shared footprint hydrocarbon profiles as well as cuticular hydrocarbons, but the profiles differed among colonies. We therefore consider that the footprint hydrocarbon profiles serve as the trail discrimination signal in L. japonicus.     

Temporal accumulation of phenylpropanoids in male fruit flies, <Emphasis Type="Italic">Bactrocera dorsalis</Emphasis> and <Emphasis Type="Italic">B. carambolae</Emphasis> (Diptera: Tephritidae) following methyl eugenol consumption

Summary. Males of dacine tephritids, Bactrocera carambolae and B. dorsalis are strongly attracted to, and compulsively feed on methyl eugenol (ME), a potent attractant for many Bactrocera species. While ME was shown to be biotransformed into phenylpropanoids, 2-allyl-4,5-dimethoxyphenol and (E)-coniferyl alcohol, and temporarily stored in the rectal gland of male B. dorsalis prior to release during courtship at dusk, B. carambolae male produces only the latter compound along with its de novo synthesized pheromone components. Both species were also shown to have different age-related response, sensitivity and consumption levels of ME. Here, we monitored and compared temporal changes in the accumulation profiles of these phenylpropanoids by the two sibling species, with male rectal glands being individually excised at different time intervals from 15 minutes to 20 days after initial ME feeding and analysed quantitatively. Results are discussed in light of plant-fruit fly co-evolution relationship.     

Trail-following behaviour in two Aphaenogaster ants

In the course of our studies on the chemical ecology of the widely distributed Mediterranean ant Aphaenogaster senilis, we found that trail following is triggered by extracts of the poison gland and Dufour’s gland. To assess the specificity of the trail pheromone, we examined whether a cross-reaction exists between trails of A. senilis and the closely related species A. iberica. Specificity seemed to differ amongst these two species, because workers of A. senilis did not follow trails of A. iberica, whereas the latter followed trails made by both species. Chemical analyses of the glandular contents reveal that Dufour’s glands of both species contain mainly alkanes and alkenes exhibiting species-specific profiles. However, differences in the poison gland content of the two species were dramatic, with A. senilis showing high amounts of alkaloids that were completely absent in A. iberica.     

Trail pheromones and Dufour gland contents in three <Emphasis Type="Italic">Camponotus</Emphasis> species (<Emphasis Type="Italic">C. castaneus,C. balzani,C. sericeiventris</Emphasis>: Formicidae,Hymenoptera)

Summary. By means of gas chromatography, gas chromatographic coupled mass spectrometry and behavioral analysis the major trail pheromone components from the hindgut of the formicine species Camponotus castaneus, C. balzani and C. sericeiventris were identified. The trail pheromone of C. castaneus is 3,5-dimethyl-6-(1-methylpropyl)- tetrahydro-2H-pyran- 2-one, and that of the other two species is 3,4-dihydro-8-hydroxy-3,5,7-trimethylisocoumarin. Although both compounds release precise trail following behavior in the respective species, the major recruitment signal in the three Camponotus species appears to be formic acid discharged from the poison gland. The composition of the Dufour gland secretions of C. castaneus and C. sericeiventris is similar, but that of the hypertrophied Dufour gland of C. balzani is very different from any other Camponotus Dufour gland content described up to date: it contains large amounts of esters, the major compound of which is octyl hexanoate, which makes up 97% a/a of the total volatiles.     

Maternal provisioning of sequestered defensive steroids by the Asian snake <Emphasis Type="Italic">Rhabdophis tigrinus</Emphasis>

Summary.   Rhabdophis tigrinus obtains defensive steroids (bufadienolides) from its diet and sequesters those compounds in specialized structures on its neck known as nuchal glands. Hatchling snakes lacking these steroids must acquire them from toads consumed as prey. Here we show that females provision bufadienolides to their offspring in amounts correlated to the quantity in their own nuchal glands; thus, chemically protected mothers produce defended offspring. Bufadienolides can be provisioned to embryos via deposition in yolk and by transfer across the egg membranes within the oviducts. Maternally provisioned bufadienolides persist in the nuchal glands of juvenile snakes from the time of hatching in late summer until the following spring, when toads of ingestible size become abundant. Therefore, maternal provisioning may provide chemical protection from predators for young R. tigrinus in the absence of dietary sources of bufadienolides.     

Combined use of pheromone trails and visual landmarks by the common garden ant Lasius niger

This study investigated the relative importance of pheromone trails and visual landmarks on the ability of Lasius niger foragers to relocate a previously used food source. Colonies formed foraging trails to a 1-M sucrose feeder. Sections of this trail were then presented back to the same colony after variable time intervals. Individual outgoing foragers were observed to determine if they walked for 15 cm in the direction of the feeder or not. On newly established pheromone trails formed by 500 ant passages, 77% of the foragers walked in the correct direction vs 31% for control foragers (no trail pheromone). Pheromone trails decayed to the control levels in 20–24 h. Trails formed with fewer ant passages (125 or 30) decayed quicker. The use of visual landmarks was investigated by using trails with outgoing foragers from the colony that established the trail, either in the same room or in a different room, with different visual landmarks, to that used during trail establishment. Approximately 20% more ants walked in the correct direction in the same room vs the different room. This difference decreased to around 10% 2 h after trail establishment, indicating that the ants in the different room were learning the new visual cues to navigate by. Our results show that visual landmarks and pheromone trails are approximately equally useful in initially guiding L. niger foragers to food locations and that these two information sources have a complementary function.     

The role of trail following in the homing of intertidal chitons: A comparison between threeAcanthopleura spp.

Three species of intertidal tropical chitons observed between 1984 and 1986, exhibit different movement patterns:Acanthopleura gemmata (Indian and Pacific Ocean) is a strict homer;A. granulata (Caribbean Sea) is mostly a ranger but sometimes shows homing behaviour;A. brevispinosa (Western Indian Ocean) alternates homing and ranging for different excursions. The three species have been studied in the field using a motographic technique to assess the importance of intra-individual trail following in homing. The fraction of the path which overlaps with earlier segments of an individual's trail is different in the three species: when returning from feeding groundsA. gemmata follows almost the whole of the outgoing path; inA. granulata the path coincidence is minimal, whileA. brevispinosa shows intermediate trail following values. InA. gemmata andA. brevispinosa trail overlap rises rapidly just after feeding, and trail following is mostly backward along the outgoing path. On the other hand,A. granulata shows a linear increase in trail coincidence with increasing path covered and the proportion of forward trail following is relatively higher. Nevertheless, all specimens of the three species which show a high homing performance invariably show a large trail following also. These findings confirm that, despite the interspecific difference in ecology and the related variation in spatial organization of foraging excursions, trail following is the basic mechanism for homing in intertidal chitons.     

A stingless bee (Melipona panamica) indicates food location without using a scent trail

The study of location specification in recruitment communication by bees has focused on two dimensions: direction and distance from the nest. Yet the third dimension, height above ground, may be significant in the tall and dense forest habitats of stingless bees. Foragers of the stingless bee Scaptotrigona postica recruit to a specific three-dimensional location by laying a scent trail. Stingless bees in the genus Melipona are thought to have a more sophisticated recruitment system that communicates distance through sounds inside the nest and direction through pointing zig-zag flights outside the nest. However, prior research on Melipona has not examined height communication or even established that foragers can recruit newcomers to a specific location. We used identical paired feeders to investigate recruitment to food in M panamica on Barro Colorado Island, Panama. We trained foragers from an observation hive to one feeder and monitored both feeders for the subsequent arrival of newcomers. We changed the relative positions of the feeders to test for correct direction, distance, and canopy-level communication. A 40-m canopy tower located inside the forest enabled us to examine canopy-level communication. We found that M. panamica foragers can recruit to a specific (1) direction, (2) distance, and (3) canopy level. To test the possibility that foragers accomplish this by means of a scent trail, we placed the colony on one shore of a small cove and trained bees over 116 m of open water to a feeder located on the opposite shore. We also placed a second feeder on this shore, equidistant from the colony but 20 m from the first feeder. Significantly more newcomers consistently arrived at the feeder visited by the foragers. Thus foragers evidently do not need a scent trail to communicate direction. Inside the nest, a forager produces pulsed sounds while visibly vibrating her wings after returning from a good food source. She is attended by other bees who cluster and hold their antennae around her, following her as she rapidly spins clockwise and counterclockwise. Locational information may be encoded in this behavior. However, foragers may also directly lead newcomers to the food source. Further experiments are planned to test for such piloting and other communication mechanisms.     

Neocembrene A, a major component of the trail-following pheromone in the genus Prorhinotermes (Insecta, Isoptera, Rhinotermitidae)

Summary. The diterpene neocembrene A or (1E,5E,9E,12R)-1,5,9-trimethyl-12-(1-methylethenyl)-1,5,9-cyclotetradecatriene, known as the trail-following pheromone of the advanced Termitidae Nasutitermitinae Nasutitermes exitiosus and Trinervitermes bettonianus, has been identified after SPME-GC/MS as the major component of the trail-following pheromone of the Rhinotermitidae Prorhinotermitinae, Prorhinotermes canalifrons and P. simplex. In all the other Rhinotermitidae studied until now, the major component of their trail pheromones is dodecatrienol ((3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol). This biochemical data further add to the anatomical and molecular characteristics that give a special status to the taxon Prorhinotermes among Rhinotermitidae. In Prorhinotermes canalifrons and P. simplex, neocembrene A was the only secretory compound specific to the sternal gland surface that could be detected after SPME. It elicited orientation as well as recruitment behavioral effects. However, the comparison of the respective biological activities triggered by neocembrene A and by sternal gland secretion suggests that minor components of the latter are acting in synergy with neocembrene A.     

Information transfer during recruitment in the ant<Emphasis Type="Italic"> Lasius niger</Emphasis> L. (Hymenoptera: Formicidae)

In this paper, we used the food-correlated search behavior observed in foraging ants returning to a previously rewarding site to study information transfer during recruitment in the ant Lasius niger. We hypothesized that, if information about the characteristics of the food is conveyed during recruitment, food-correlated search tactics should also be observed in recruited workers. Our results show that the characteristics of the trajectories of recruited workers are comparable to those of scout ants returning to a site or prior food find and depend more on the type (prey/sugar) than on the quality (sugar concentration) of the food discovered by the scouts. Independent of sugar concentration, workers recruited to a source of sugar search with a greater sinuosity than workers recruited to a prey. Experimental manipulation of the recruitment signals (chemical trail and contact between ants) shows that the trail pheromone laid down by recruiting ants does not play a role in the modification of trajectory sinuosity. This change appears to be most likely triggered by a direct perception of the residue of sugar smeared on the body of the recruiting workers coming back to the nest.Communicated by J. Heinze     

Species-specificity in trail pheromones and Dufour's gland contents ofCamponotus atriceps andC. floridanus (Hymenoptera: Formicidae)

Summary By means of gas chromatography, gas chromatographic coupled mass spectrometry, trail-following experiments and electrophysiological recordings from worker antennae, the major trail pheromone components from the hindgut of the formicine speciesCamponotus atriceps andC. floridanus were identified as 3,5-dimethyl-6-(1-methylpropyl)-tetrahydropyran-2-one and nerolic acid, respectively. The Dufour's gland contents of both species, investigated by gas chromatographic coupled mass spectrometry, show significant differences.Pheromones 104: Janssen E, Übler E, Bauriegel L, Kern F, Bestmann H-J, Attygalle AB, Steghaus-Kovacs S, Maschwitz U: Trail pheromone of the Ponerinae antLeptogenys peuqueti (Hymenoptera: Formicidae): a multicomponent mixture of related compounds     

Identification and biological activity of sex pheromone components from females of the plum moth Illiberis rotundata Jordan (Lepidoptera: Zygaenidae: Procridinae)

The plum moth, Illiberis rotundata Jordan (Lepidoptera: Zygaenidae: Procridinae), is a pest of orchards in Japan and China. Few chemical ecological studies have been directed towards the Zygaenidae and particularly the Procridinae. To investigate the sex pheromone of this species, extracts of pheromone glands from adult female I. rotundata were analyzed by coupled gas chromatography-electroantennography (GC-EAG) and coupled gas chromatography-mass spectrometry (GC-MS). Whilst GC-EAG on male moths showed an active peak, identified as 2-butyl (7Z)-dodecenoate, GC-MS also revealed the presence of the homologue 2-butyl (9Z)-tetradecenoate. Electroantennographic investigations, as well as field tests, strongly suggested the natural compounds to have the (R)-configuration at the stereogenic centre. Field results demonstrate 0.2 mg of a 1:1-mixture of (2R)-butyl (7Z)-dodecenoate and (2R)-butyl (9Z)-tetradecenoate to be a powerful lure that may be used in pest control measures against I. rotundata. The chemical structures of the new pheromone components show the same features as those of other zygaenid species: unsaturated fatty acids esterified with a short chain chiral alcohol. This is the first example of a two-component blend constituting the pheromone of a procridinid species.     

Mate preferences in <Emphasis Type="Italic">Drosophila</Emphasis> infected with <Emphasis Type="Italic">Wolbachia</Emphasis>?

The maternally inherited bacterium Wolbachia pipientis generates strong reproductive incompatibilities between uninfected females and infected males (cytoplasmic incompatibility), significantly reducing both female and male reproductive success. Such fitness costs are thought to place selective pressure on hosts to evolve pre-copulatory preferences for mating with compatible mates, thereby enabling them to avoid the reproductive incompatibilities associated with Wolbachia. Therefore, uninfected females are predicted to prefer mating with uninfected males, whereas infected males are predicted to prefer mating with infected females. Despite these predictions, previous investigations of pre-copulatory mate preferences in Wolbachia-manipulated Drosophila have not found evidence of female preference for uninfected or compatible males. However, none of these studies utilised a design where focal individuals are provided with a simple choice in a relatively non-competitive situation. We examined both female and male pre-copulatory mate preference based on mate infection status in Drosophila simulans and D. melanogaster using simple choice assays involving between 30–50 replicates per treatment. Although we found no evidence of female pre-copulatory mate preferences in either species, male D. simulans exhibited some preference for mating with females of the same infection status. However, this preference was not evident when we repeated the experiment to confirm this finding. Consequently, we conclude that neither male nor female D. melanogaster and D. simulans exhibit significant Wolbachia-associated pre-copulatory mate preferences.     

The chemical basis for trail recognition in Lasius nipponensis (Hymenoptera: Formicidae)

Summary. In this paper we report on the chemical basis for trail recognition in Lasius nipponensis. On and near trails in the field, workers became aggressive against conspecific intruders and succeeded in protective contests, while intruders usually avoided confrontation. Such asymmetric interactions were also observed in the laboratory when two non-nestmate workers were sequentially placed in a clean glass dish, while mutual aggression was observed when they were placed simultaneously. Asymmetric aggression was also observed when the workers were placed in a dish previously conditioned by other workers, or when the dish was treated with the hexane rinse of the conditioned dish. The rinse contained a series of hydrocarbons, in which components and proportion were almost identical to those of the cuticular hydrocarbons of the workers, except for a lack of n-alkanes. Amounts of the hydrocarbons that remained on the dish were very small when all the tarsi of the workers were incapacitated. We therefore consider that the hydrocarbons are not adhesion of the cuticular hydrocarbons but secretion from the tarsi of workers, and serve as a key signal for trail recognition in this ant.     

Structure and function of the eversible glands of the aquatic firefly <Emphasis Type="Italic">Luciola leii</Emphasis> (Coleoptera: Lampyridae)

Summary. When attacked or otherwise disturbed, larvae of the aquatic firefly Luciola leii display fork-shaped glands laterally from the meso- and metathorax as well as each abdominal segment. Glandular eversion is accompanied by a strong pine oil-like odour, thanatosis and glowing from paired larval light organs. Observations by SEM and TEM revealed that there are numerous, almost spherical protuberances, measuring 9 μm in diameter on the surface of each gland. Each protuberance is connected to a well developed secretory cell via a 0.1 μm thick and 0.2 μm long stalk and bears three to six 7 μm long spines on its apex. The convoluted glandular walls measure 0.2 μm. The cytoplasm of the secretory cells is characterized by the presence of numerous mitochondria and an extensive system of cisternae and tubular endoplasmic reticulum. Preliminary GC-MS analysis of the glandular secretion revealed two volatile terpenoids: terpinolene and γ-terpinene. Choice and no-choice bioassays involving fish and ants as well as other predators demonstrate that the secretions serve as an effective deterrent against a range of ecologically relevant enemies. The larval postural adjustments, light emission, everted glands, and glandular release of distasteful or repellent compounds, appear to function as a multi-modal, aposematic larval defence. The tandem evolution of glandular secretions and conspicuousness in larval fireflies could partially explain their successful radiation into both aquatic and terrestrial habitats.